Compositions and methods for the treatment of viral infections

ABSTRACT

Compositions and methods for the treatment of viral infections include conjugates containing inhibitors of viral neuraminidase (e.g., zanamivir, peramivir, or analogs thereof) linked to an Fc monomer, an Fc domain, and Fc-binding peptide, an albumin protein, or albumin-binding peptide. In particular, conjugates can be used in the treatment of viral infections (e.g., influenza viral infections).

BACKGROUND

The need for novel antiviral treatments for influenza is significant andespecially critical in the medical field. Influenza virus, the causativeagent of influenza, or the flu, is responsible for three to five millioncases of severe illness annually, and approximately 500,000 deathsworldwide. While most people recover completely from influenza in aboutone to two weeks, others develop life-threatening complications, such aspneumonia. Thus, influenza can be deadly, especially for the young, old,or chronically ill. People with weak or compromised immune systems, suchas people with advanced HIV infection or transplant patients, whoseimmune systems are medically suppressed to prevent transplant organrejection, are at greater risk for complications relating to influenza.Pregnant women and young children are also at a high risk forcomplications.

The development of antiviral treatments for influenza has been acontinuing challenge. Several influenza antiviral agents have beenapproved for use in the clinic, and these agents play important roles inmodulating disease severity and controlling pandemics while vaccines areprepared. However, drug-resistant strains have emerged to the mostcommonly used inhibitors.

Influenza antiviral agents largely target proteins presented on thesurface of the influenza virus particle. The envelope of the influenzavirus contains two immunodominant glycoproteins, hemagglutinin andneuraminidase, that play key roles in viral infection and spread.Hemagglutinin effects attachment of the virus to the host cell throughits interaction with surface sialic acids, thereby initiating entry.Neuraminidase is an exo-glycosidase enzyme that cleaves sialic acids(terminal neuraminic acid residues) from glycan structures on thesurface of infected host cells, releasing progeny viruses and allowingthe spread of the virus from the host cell to uninfected surroundingcells. Inhibition of neuraminidase therefore serves as a pharmacologicaltarget for antiviral drugs. Viral neuraminidase inhibitors used toreduce viral spread have been identified, including oseltamivir(Tamiflu™), zanamivir (Relenza™), and peramivir (Rapivab™).

However, influenza in transplant recipients remains characterized byprolonged viral shedding, increasing the likelihood of developing drugresistant strains. New, more effective therapies for treating influenzaare needed.

SUMMARY

The disclosure relates to conjugates, compositions, and methods forinhibiting viral growth, and methods for the treatment of viralinfections. In particular, such conjugates contain monomers or dimers ofa moiety that inhibits influenza virus neuraminidase (e.g., zanamivir,peramivir, or analogs thereof) conjugated to Fc monomers, Fc domains,Fc-binding peptides, albumin proteins, or albumin protein-bindingpeptides. The neuraminidase inhibitor (e.g., zanamivir, peramivir, oranalogs thereof) in the conjugates targets neuraminidase on the surfaceof the viral particle. The Fc monomers or Fc domains in the conjugatesbind to FcγRs (e.g., FcRn, FcγRI, FcγRIIa, FcγRIIc, FcγRIIIa, andFcγRIIIb) on immune cells, e.g., neutrophils, to activate phagocytosisand effector functions, such as antibody-dependent cell-mediatedcytotoxicity (ADCC), thus leading to the engulfment and destruction ofviral particles by immune cells and further enhancing the antiviralactivity of the conjugates. The albumin or albumin-binding peptide mayextend the half-life of the conjugate, for example, by binding ofalbumin to the recycling neonatal Fc receptor. Such compositions areuseful in methods for the inhibition of viral growth and in methods forthe treatment of viral infections, such as those caused by an influenzavirus A, influenza virus B and influenza virus C.

In one aspect, the disclosure features a conjugate described by formula(1):

wherein each A₁ and each A₂ is independently selected from any one offormulas (A-I)-(A-XII):

wherein R₁ is selected from —OH, —NH₂, —NHC(═NH)NH₂, and —NHC(═NH)NHR₆;R₂ and R₃ are each independently selected from —H, —OH, —F, —Cl, and—Br; R₄ is selected from —CO₂H, —P(═O)(OH)₂, —SO₃H; R₅ is selected from—COCH₃, —COCF₃, —SO₂CH₃; X is selected from —O— and —S—; Y is selectedfrom

R₆ is selected from

R₇ is selected from H, C1-C20 alkyl, C3-C20 cycloalkyl, C3-C20heterocycloalkyl; C5-C15 aryl, and C2-C15 heteroaryl; R₈ is selectedfrom C3-C20 heterocycloalkyl, C5-C15 aryl, and C2-C15 heteroaryl; each Ecomprises an Fc domain monomer (e.g., an Fc domain monomer having thesequence of any one of SEQ ID NOs: 1-68); L in each A₁-L-A₂ is a linkercovalently attached to a sulfur atom of a hinge cysteine in each E andto each of A₁ and A₂; T is an integer from 1 to 20 (e.g., 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), and the twosquiggly lines connected to the two Es indicate that each A₁-L-A₂ iscovalently attached (e.g., byway of a covalent bond or a linker) to apair of sulfur atoms of two hinge cysteines in the two Es, or apharmaceutically acceptable salt thereof. When T is greater than 1(e.g., T is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, or 20), each A₁-L-A₂ may be independently selected (e.g.,independently selected from any of the A₁-L-A₂ structures describedherein).

In another aspect, the disclosure features a conjugate described byformula (1):

wherein each A₁ and each A₂ is independently selected from any one offormulas (A-I)-(A-V):

wherein R₁ is selected from —OH, —NH₂, —NHC(═NH)NH₂, and —NHC(═NH)NHR₆;R₂ and R₃ are each independently selected from —H, —OH, —F, —Cl, and—Br; R₄ is selected from —CO₂H, —P(═O)(OH)₂, —SO₃H; R₅ is selected from—COCH₃, —COCF₃, —SO₂CH₃; X is selected from —O— and —S—; Y is selectedfrom

R₆ is selected from

R₇ is selected from H, C1-C20 alkyl, C3-C20 cycloalkyl, C3-C20heterocycloalkyl; C5-C15 aryl, and C2-C15 heteroaryl; R₈ is selectedfrom C3-C20 heterocycloalkyl, C5-C15 aryl, and C2-C15 heteroaryl; each Ecomprises an Fc domain monomer (e.g., an Fc domain monomer having thesequence of any one of SEQ ID NOs: 1-68); L in each A₁-L-A₂ is a linkercovalently attached to a sulfur atom of a hinge cysteine in each E andto each of A₁ and A₂; T is an integer from 1 to 20 (e.g., 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), and the twosquiggly lines connected to the two Es indicate that each A₁-L-A₂ iscovalently attached (e.g., byway of a covalent bond or a linker) to apair of sulfur atoms of two hinge cysteines in the two Es, or apharmaceutically acceptable salt thereof. When T is greater than 1(e.g., T is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, or 20), each A₁-L-A₂ may be independently selected (e.g.,independently selected from any of the A₁-L-A₂ structures describedherein).

In another aspect, the disclosure features a conjugate described byformula (1):

wherein each A₁ and each A₂ is independently selected from any one offormulas (A-VI)-(A-IX):

wherein R₁ is selected from —OH, —NH₂, —NHC(═NH)NH₂, and —NHC(═NH)NHR₆;R₂ and R₃ are each independently selected from —H, —OH, —F, —Cl, and—Br; R₄ is selected from —CO₂H, —P(═O)(OH)₂, —SO₃H; R₅ is selected from—COCH₃, —COCF₃, —SO₂CH₃; X is selected from —O— and —S—; Y is selectedfrom

R₆ is selected from

R₇ is selected from H, C1-C20 alkyl, C3-C20 cycloalkyl, C3-C20heterocycloalkyl; C5-C15 aryl, and C2-C15 heteroaryl; R₈ is selectedfrom C3-C20 heterocycloalkyl, C5-C15 aryl, and C2-C15 heteroaryl; each Ecomprises an Fc domain monomer (e.g., an Fc domain monomer having thesequence of any one of SEQ ID NOs: 1-68); L in each A₁-L-A₂ is a linkercovalently attached to a sulfur atom of a hinge cysteine in each E andto each of A₁ and A₂; T is an integer from 1 to 20 (e.g., 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), and the twosquiggly lines connected to the two Es indicate that each A₁-L-A₂ iscovalently attached (e.g., byway of a covalent bond or a linker) to apair of sulfur atoms of two hinge cysteines in the two Es, or apharmaceutically acceptable salt thereof. When T is greater than 1(e.g., T is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, or 20), each A₁-L-A₂ may be independently selected (e.g.,independently selected from any of the A₁-L-A₂ structures describedherein). In another aspect, the invention features a conjugate describedby formula (2):

wherein each A₁ is independently selected from any one of formulas(A-I)-(A-XII):

wherein R₁ is selected from —OH, —NH₂, —NHC(═NH)NH₂, and —NHC(═NH)NHR₆;R₂ and R₃ are each independently selected from —H, —OH, —F, —Cl, and—Br; R₄ is selected from —CO₂H, —P(═O)(OH)₂, —SO₃H; R₅ is selected from—COCH₃, —COCF₃, —SO₂CH₃; X is selected from —O— and —S—; Y is selectedfrom

R₆ is selected from

R₇ is selected from H, C1-C20 alkyl, C3-C20 cycloalkyl, C3-C20heterocycloalkyl; C5-C15 aryl, and C2-C15 heteroaryl; R₈ is selectedfrom C3-C20 heterocycloalkyl, C5-C15 aryl, and C2-C15 heteroaryl; each Ecomprises an Fc domain monomer (e.g., an Fc domain monomer having thesequence of any one of SEQ ID NOs: 1-68); L in each L-A₁ is a linkercovalently attached to a sulfur atom in a hinge cysteine in E and to A₁;T is an integer from 1 to 20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, or 20), and the two squiggly linesconnected to the two sulfur atoms indicate that each L-A₁ is covalently(e.g., by way of a covalent bond or a linker) attached to a pair ofsulfur atoms of two hinge cysteines in the two Es, or a pharmaceuticallyacceptable salt thereof. When T is greater than 1 (e.g., T is 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), each A₁may be independently selected from any one of formulas (A-I)-(A-XII).

In another aspect, the invention features a conjugate described byformula (2):

wherein each A₁ is independently selected from any one of formulas(A-I)-(A-V):

wherein R₁ is selected from —OH, —NH₂, —NHC(═NH)NH₂, and —NHC(═NH)NHR₆;R₂ and R₃ are each independently selected from —H, —OH, —F, —Cl, and—Br; R₄ is selected from —CO₂H, —P(═O)(OH)₂, —SO₃H; R₅ is selected from—COCH₃, —COCF₃, —SO₂CH₃; X is selected from —O— and —S—; Y is selectedfrom

R₆ is selected from

R₇ is selected from H, C1-C20 alkyl, C3-C20 cycloalkyl, C3-C20heterocycloalkyl; C5-C15 aryl, and C2-C15 heteroaryl; R₈ is selectedfrom C3-C20 heterocycloalkyl, C5-C15 aryl, and C2-C15 heteroaryl; each Ecomprises an Fc domain monomer (e.g., an Fc domain monomer having thesequence of any one of SEQ ID NOs: 1-68); L in each L-A₁ is a linkercovalently attached to a sulfur atom in a hinge cysteine in E and to A₁;T is an integer from 1 to 20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, or 20), and the two squiggly linesconnected to the two sulfur atoms indicate that each L-A₁ is covalentlyattached (e.g., by way of a covalent bond or a linker) to a pair ofsulfur atoms of two hinge cysteines in the two Es, or a pharmaceuticallyacceptable salt thereof. When T is greater than 1 (e.g., T is 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), each A₁may be independently selected from any one of formulas (A-I)-(A-V).

In another aspect, the invention features a conjugate described byformula (2):

wherein each A₁ is independently selected from any one of formulas(A-VI)-(A-IX):

wherein R₁ is selected from —OH, —NH₂, —NHC(═NH)NH₂, and —NHC(═NH)NHR₆;R₂ and R₃ are each independently selected from —H, —OH, —F, —Cl, and—Br; R₄ is selected from —CO₂H, —P(═O)(OH)₂, —SO₃H; R₅ is selected from—COCH₃, —COCF₃, —SO₂CH₃; X is selected from —O— and —S—; Y is selectedfrom

R₆ is selected from

R₇ is selected from H, C1-C20 alkyl, C3-C20 cycloalkyl, C3-C20heterocycloalkyl; C5-C15 aryl, and C2-C15 heteroaryl; R₈ is selectedfrom C3-C20 heterocycloalkyl, C5-C15 aryl, and C2-C15 heteroaryl; each Ecomprises an Fc domain monomer (e.g., an Fc domain monomer having thesequence of any one of SEQ ID NOs: 1-68); L in each L-A₁ is a linkercovalently attached to a sulfur atom in a hinge cysteine in E and to A₁;T is an integer from 1 to 20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, or 20), and the two squiggly linesconnected to the two sulfur atoms indicate that each L-A₁ is covalentlyattached (e.g., by way of a covalent bond or a linker) to a pair ofsulfur atoms of two hinge cysteines in the two Es, or a pharmaceuticallyacceptable salt thereof. When T is greater than 1 (e.g., T is 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), each A₁may be independently selected from any one of formulas (A-VI)-(A-IX).

In some embodiments of any of the foregoing embodiments, each E includesan Fc domain monomer having the sequence of any one of SEQ ID NOs: 1-68.

In some embodiments, each E comprises the sequence

(SEQ ID NO: 10) MVRSDKTHTCPPCPPC*KC*PAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKHHHHHH.

In some embodiments, each E comprises the sequence

(SEQ ID NO: 11) MVRSDKTHTCPPCPPC*KC*PAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

In some embodiments, at least one of the pair of sulfur atoms is thesulfur atom corresponding to (e.g., the sulfur atom of) a hinge cysteineof SEQ ID NO: 10 or SEQ ID NO: 11, i.e., Cys10, Cys13, Cys16, or Cys18of SEQ ID NO: 10 or SEQ ID NO: 11. In some embodiments, the pair ofsulfur atoms are the sulfur atoms corresponding to (e.g., the sulfuratoms of) Cys10 and Cys13 in SEQ ID NO: 10 or SEQ ID NO: 11, Cys10 andCys16 in SEQ ID NO: 10 or SEQ ID NO: 11, Cys 30 and Cys18 in SEQ ID NO:10 or SEQ ID NO: 11, Cys13 and Cys 36 in SEQ ID NO: 10 or SEQ ID NO: 11,Cys13 and Cys 38 in SEQ ID NO: 10 or SEQ ID NO: 11, and/or Cys 36 andCys 38 in SEQ ID NO: 10 or SEQ ID NO: 11.

In some embodiments, when T is 2, the pair of sulfur atoms are (e.g.,the sulfur atoms corresponding to) Cys10 and Cys13 in SEQ ID NO: 10 orSEQ ID NO: 11 or Cys 36 and Cys 38 in SEQ ID NO: 10 or SEQ ID NO: 11.

In some embodiments, the pair of sulfur atoms include one sulfur atom ofa cysteine from each E, i.e., L-A along with the sulfur atoms to whichit is attached forms a bridge between two Fc domains (e.g., two Fcdomains comprising the sequence of SEQ ID NO: 10 or SEQ ID NO: 11). Insome embodiments, the pair of sulfur atoms are the sulfur atomcorresponding to (e.g., the sulfur atom of) Cys10 of SEQ ID NO: 10 orSEQ ID NO: 11 from one E and the sulfur atom corresponding to (e.g., thesulfur atom of) Cys10 of SEQ ID NO: 10 or SEQ ID NO: 11 from another E.In some embodiments, the pair of sulfur atoms are the sulfur atomcorresponding to (e.g., the sulfur atom of) Cys13 of SEQ ID NO: 10 orSEQ ID NO: 11 from one E and the sulfur atom corresponding to (e.g., thesulfur atom of) Cys13 of SEQ ID NO: 10 or SEQ ID NO: 11 from another E.In some embodiments, the pair of sulfur atoms are the sulfur atomcorresponding to (e.g., the sulfur atom of) Cys16 of SEQ ID NO: 10 orSEQ ID NO: 11 from one E and the sulfur atom corresponding to (e.g., thesulfur atom of) Cys16 of SEQ ID NO: 10 or SEQ ID NO: 11 from another E.In some embodiments, the pair of sulfur atoms are the sulfur atomcorresponding to (e.g., the sulfur atom of) Cys18 of SEQ ID NO: 10 orSEQ ID NO: 11 from one E and the sulfur atom corresponding to (e.g., thesulfur atom of) Cys18 of SEQ ID NO: 10 or SEQ ID NO: 11 from another E.

In some embodiments, when T is 2, the pairs of sulfur atoms are thesulfur atom corresponding to (e.g., the sulfur atom of) Cys10 of SEQ IDNO: 10 or SEQ ID NO: 11 from one E and the sulfur atom corresponding to(e.g., the sulfur atom of) Cys10 of SEQ ID NO: 10 or SEQ ID NO: 11 fromanother E and the sulfur atom corresponding to (e.g., the sulfur atomof) Cys13 of SEQ ID NO: 10 or SEQ ID NO: 11 from one E and the sulfuratom corresponding to (e.g., the sulfur atom of) Cys13 of SEQ ID NO: 10or SEQ ID NO: 11 from another E. In some embodiments, when T is 2, thepairs of sulfur atoms are the sulfur atom corresponding to (e.g., thesulfur atom of) Cys10 of SEQ ID NO: 10 or SEQ ID NO: 11 from one E andthe sulfur atom corresponding to (e.g., the sulfur atom of) Cys10 of SEQID NO: 10 or SEQ ID NO: 11 from another E and the sulfur atomcorresponding to (e.g., the sulfur atom of) Cys16 of SEQ ID NO: 10 orSEQ ID NO: 11 from one E and the sulfur atom corresponding to (e.g., thesulfur atom of) Cys16 of SEQ ID NO: 10 or SEQ ID NO: 11 from another E.In some embodiments, when T is 2, the pairs of sulfur atoms are thesulfur atom corresponding to (e.g., the sulfur atom of) Cys10 of SEQ IDNO: 10 or SEQ ID NO: 11 from one E and the sulfur atom corresponding to(e.g., the sulfur atom of) Cys10 of SEQ ID NO: 10 or SEQ ID NO: 11 fromanother E and the sulfur atom corresponding to (e.g., the sulfur atomof) Cys18 of SEQ ID NO: 10 or SEQ ID NO: 11 from one E and the sulfuratom corresponding to (e.g., the sulfur atom of) Cys18 of SEQ ID NO: 10or SEQ ID NO: 11 from another E.

In some embodiments, when T is 2, the pairs of sulfur atoms are thesulfur atom corresponding to (e.g., the sulfur atom of) Cys13 of SEQ IDNO: 10 or SEQ ID NO: 11 from one E and the sulfur atom corresponding to(e.g., the sulfur atom of) Cys13 of SEQ ID NO: 10 or SEQ ID NO: 11 fromanother E and the sulfur atom corresponding to (e.g., the sulfur atomof) Cys16 of SEQ ID NO: 10 or SEQ ID NO: 11 from one E and the sulfuratom corresponding to (e.g., the sulfur atom of) Cys16 of SEQ ID NO: 10or SEQ ID NO: 11 from another E. In some embodiments, when T is 2, thepairs of sulfur atoms are the sulfur atom corresponding to (e.g., thesulfur atom of) Cys13 of SEQ ID NO: 10 or SEQ ID NO: 11 from one E andthe sulfur atom corresponding to (e.g., the sulfur atom of) Cys13 of SEQID NO: 10 or SEQ ID NO: 11 from another E and the sulfur atomcorresponding to (e.g., the sulfur atom of) Cys18 of SEQ ID NO: 10 orSEQ ID NO: 11 from one E and the sulfur atom corresponding to (e.g., thesulfur atom of) Cys18 of SEQ ID NO: 10 or SEQ ID NO: 11 from another E.

In some embodiments, when T is 2, the pairs of sulfur atoms are thesulfur atom corresponding to (e.g., the sulfur atom of) Cys16 of SEQ IDNO: 10 or SEQ ID NO: 11 from one E and the sulfur atom corresponding to(e.g., the sulfur atom of) Cys16 of SEQ ID NO: 10 or SEQ ID NO: 11 fromanother E and the sulfur atom corresponding to (e.g., the sulfur atomof) Cys18 of SEQ ID NO: 10 or SEQ ID NO: 11 from one E and the sulfuratom corresponding to (e.g., the sulfur atom of) Cys18 of SEQ ID NO: 10or SEQ ID NO: 11 from another E.

In some embodiments, when T is 3, the pairs of sulfur atoms are thesulfur atom corresponding to (e.g., the sulfur atom of) Cys10 of SEQ IDNO: 10 or SEQ ID NO: 11 from one E and the sulfur atom corresponding to(e.g., the sulfur atom of) Cys10 of SEQ ID NO: 10 or SEQ ID NO: 11 fromanother E; the sulfur atom corresponding to (e.g., the sulfur atom of)Cys13 of SEQ ID NO: 10 or SEQ ID NO: 11 from one E and the sulfur atomcorresponding to (e.g., the sulfur atom of) Cys13 of SEQ ID NO: 10 orSEQ ID NO: 11 from another E; and the sulfur atom corresponding to(e.g., the sulfur atom of) Cys16 of SEQ ID NO: 10 or SEQ ID NO: 11 fromone E and the sulfur atom corresponding to (e.g., the sulfur atom of)Cys16 of SEQ ID NO: 10 or SEQ ID NO: 11 from another E. In someembodiments, when T is 3, the pairs of sulfur atoms are the sulfur atomcorresponding to (e.g., the sulfur atom of) Cys10 of SEQ ID NO: 10 orSEQ ID NO: 11 from one E and the sulfur atom corresponding to (e.g., thesulfur atom of) Cys10 of SEQ ID NO: 10 or SEQ ID NO: 11 from another E;the sulfur atom corresponding to (e.g., the sulfur atom of) Cys13 of SEQID NO: 10 or SEQ ID NO: 11 from one E and the sulfur atom correspondingto (e.g., the sulfur atom of) Cys13 of SEQ ID NO: 10 or SEQ ID NO: 11from another E; and the sulfur atom corresponding to (e.g., the sulfuratom of) Cys18 of SEQ ID NO: 10 or SEQ ID NO: 11 from one E and thesulfur atom corresponding to (e.g., the sulfur atom of) Cys18 of SEQ IDNO: 10 or SEQ ID NO: 11 from another E. In some embodiments, when T is3, the pairs of sulfur atoms are the sulfur atom corresponding to (e.g.,the sulfur atom of) Cys10 of SEQ ID NO: 10 or SEQ ID NO: 11 from one Eand the sulfur atom corresponding to (e.g., the sulfur atom of) Cys10 ofSEQ ID NO: 10 or SEQ ID NO: 11 from another E; the sulfur atomcorresponding to (e.g., the sulfur atom of) Cys18 of SEQ ID NO: 10 orSEQ ID NO: 11 from one E and the sulfur atom corresponding to (e.g., thesulfur atom of) Cys18 of SEQ ID NO: 10 or SEQ ID NO: 11 from another E;and the sulfur atom corresponding to (e.g., the sulfur atom of) Cys16 ofSEQ ID NO: 10 or SEQ ID NO: 11 from one E and the sulfur atomcorresponding to (e.g., the sulfur atom of) Cys16 of SEQ ID NO: 10 orSEQ ID NO: 11 from another E. In some embodiments, when T is 3, thepairs of sulfur atoms are the sulfur atom corresponding to (e.g., thesulfur atom of) Cys13 of SEQ ID NO: 10 or SEQ ID NO: 11 from one E andthe sulfur atom corresponding to (e.g., the sulfur atom of) Cys13 of SEQID NO: 10 or SEQ ID NO: 11 from another E; the sulfur atom correspondingto (e.g., the sulfur atom of) Cys18 of SEQ ID NO: 10 or SEQ ID NO: 11from one E and the sulfur atom corresponding to (e.g., the sulfur atomof) Cys18 of SEQ ID NO: 10 or SEQ ID NO: 11 from another E; and thesulfur atom corresponding to (e.g., the sulfur atom of) Cys16 of SEQ IDNO: 10 or SEQ ID NO: 11 from one E and the sulfur atom corresponding to(e.g., the sulfur atom of) Cys16 of SEQ ID NO: 10 or SEQ ID NO: 11 fromanother E.

In some embodiments, when T is 3, the pairs of sulfur atoms are thesulfur atom corresponding to (e.g., the sulfur atom of) Cys10 of SEQ IDNO: 10 or SEQ ID NO: 11 from one E and the sulfur atom corresponding to(e.g., the sulfur atom of) Cys10 of SEQ ID NO: 10 or SEQ ID NO: 11 fromanother E; the sulfur atom corresponding to (e.g., the sulfur atom of)Cys13 of SEQ ID NO: 10 or SEQ ID NO: 11 from one E and the sulfur atomcorresponding to (e.g., the sulfur atom of) Cys13 of SEQ ID NO: 10 orSEQ ID NO: 11 from another E; the sulfur atom corresponding to (e.g.,the sulfur atom of) Cys16 of SEQ ID NO: 10 or SEQ ID NO: 11 from one Eand the sulfur atom corresponding to (e.g., the sulfur atom of) Cys16 ofSEQ ID NO: 10 or SEQ ID NO: 11 from another E; and the sulfur atomcorresponding to (e.g., the sulfur atom of) Cys18 of SEQ ID NO: 10 orSEQ ID NO: 11 from one E and the sulfur atom corresponding to (e.g., thesulfur atom of) Cys18 of SEQ ID NO: 10 or SEQ ID NO: 11 from another E.

In some embodiments, the conjugate has the structure:

wherein each of a, b, c, and d is, independently, 0 or 1 and whereinwhen a, b, c, or d is 0, the two sulfur atoms form a disulfide bond.

In some embodiments, a is 1 and b, c, and d are 0. In some embodiments,a and b are 1 and c and d are 0. In some embodiments, a and c are 1 andb and d are 0. In some embodiments, a and d are 1 and b and c are 0. Insome embodiments, a, b, and c are 1 and d is 0. In some embodiments, a,b, and d are 1 and c is 0. In some embodiments, a, c, and d are 1 and bis 0. In some embodiments, b and c are 1 and a and d are 0. In someembodiments, b and d are 1 and a and c are 0. In some embodiments, b, c,and d are 1 and a is 0. In some embodiments, c and d are 1 and a and bare 0. In some embodiments, a, b, c, and d are 1.

In some embodiments, each E comprises the sequence

(SEQ ID NO: 4) MVRSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

In some embodiments, each E comprises the sequence

(SEQ ID NO: 33) MVRSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

In some embodiments, at least one of the pair of sulfur atoms is thesulfur atom corresponding to (e.g., the sulfur atom of) a hinge cysteineof SEQ ID NO: 4 or SEQ ID NO: 33, i.e., Cys10 and/or Cys13. In someembodiments, the pair of sulfur atoms are the sulfur atoms correspondingto (e.g., the sulfur atoms of) Cys10 and Cys13 in SEQ ID NO: 4 or SEQ IDNO: 33.

In some embodiments, the pair of sulfur atoms include one sulfur atom ofa cysteine from each E, i.e., L-A along with the sulfur atoms to whichit is attached forms a bridge between two Fc domains (e.g., two Fcdomains comprising the sequence of SEQ ID NO: 4 or SEQ ID NO: 33). Insome embodiments, the pair of sulfur atoms are the sulfur atomcorresponding to (e.g., the sulfur atom of) Cys10 of SEQ ID NO: 4 or SEQID NO: 33 from one E and the sulfur atom corresponding to (e.g., thesulfur atom of) Cys10 of SEQ ID NO: 4 or SEQ ID NO: 33 from another E.In some embodiments, the pair of sulfur atoms are the sulfur atomcorresponding to (e.g., the sulfur atom of) Cys13 of SEQ ID NO: 4 or SEQID NO: 33 from one E and the sulfur atom corresponding to (e.g., thesulfur atom of) Cys13 of SEQ ID NO: 4 or SEQ ID NO: 33 from another E.In some embodiments, when T is 2, the pairs of sulfur atoms are thesulfur atom corresponding to (e.g., the sulfur atom of) Cys10 of SEQ IDNO: 4 or SEQ ID NO: 33 from one E and the sulfur atom corresponding to(e.g., the sulfur atom of) Cys10 of SEQ ID NO: 4 or SEQ ID NO: 33 fromanother E and the sulfur atom corresponding to (e.g., the sulfur atomof) Cys13 of SEQ ID NO: 4 or SEQ ID NO: 33 from one E and the sulfuratom corresponding to (e.g., the sulfur atom of) Cys13 of SEQ ID NO: 4or SEQ ID NO: 33 from another E.

In some embodiments, the conjugate has the structure:

wherein each of a and b is, independently, 0 or 1 and wherein when a orb is 0, the two sulfur atoms form a disulfide bond. In some embodiments,a is 1 and b is 0. In some embodiments, a is 0 and b is 1. In someembodiments, a and b are 1.

In some embodiments, each E comprises the sequence

(SEQ ID NO: 8) MVRSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKHHHHHH.

In some embodiments, at least one of the pair of sulfur atoms is thesulfur atom corresponding to (e.g., the sulfur atom of) a hinge cysteineof SEQ ID NO: 8, i.e., Cys10 and/or Cys13. In some embodiments, the pairof sulfur atoms are the sulfur atoms corresponding to (e.g., the sulfuratoms of) Cys10 and Cys13 in SEQ ID NO: 8.

In some embodiments, the pair of sulfur atoms include one sulfur atom ofa cysteine from each E, i.e., L-A along with the sulfur atoms to whichit is attached forms a bridge between two Fc domains (e.g., two Fcdomains comprising the sequence of SEQ ID NO: 8). In some embodiments,the pair of sulfur atoms are the sulfur atom corresponding to (e.g., thesulfur atom of) Cys10 of SEQ ID NO: 8 from one E and the sulfur atomcorresponding to (e.g., the sulfur atom of) Cys10 of SEQ ID NO: 8 fromanother E. In some embodiments, the pair of sulfur atoms are the sulfuratom corresponding to (e.g., the sulfur atom of) Cys13 of SEQ ID NO: 8from one E and the sulfur atom corresponding to (e.g., the sulfur atomof) Cys13 of SEQ ID NO: 8 from another E. In some embodiments, when T is2, the pairs of sulfur atoms are the sulfur atom corresponding to (e.g.,the sulfur atom of) Cys10 of SEQ ID NO: 8 from one E and the sulfur atomcorresponding to (e.g., the sulfur atom of) Cys10 of SEQ ID NO: 8 fromanother E and the sulfur atom corresponding to (e.g., the sulfur atomof) Cys13 of SEQ ID NO: 8 from one E and the sulfur atom correspondingto (e.g., the sulfur atom of) Cys13 of SEQ ID NO: 8 from another E.

In some embodiments, the conjugate has the structure:

wherein each of a and b is, independently, 0 or 1 and wherein when a orb is 0, the two sulfur atoms form a disulfide bond. In some embodiments,a is 1 and b is 0. In some embodiments, a is 0 and b is 1. In someembodiments, a and b are 1.

In another aspect, the invention also features a population ofconjugates described in any of the previous aspects, in which theaverage value of T is 1 to 20 (e.g., the average value of T is 1 to 2, 1to 3, 1 to 4, 1 to 5, 5 to 10, 10 to 15, or 15 to 20). In someembodiments, the average value of T is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.

In another aspect, the invention features a conjugate described byformula (3):

wherein each A₁ and each A₂ is independently selected from any one offormulas (A-I)-(A-VII):

wherein R₁ is selected from —OH, —NH₂, —NHC(═NH)NH₂, and —NHC(═NH)NHR₆;R₂ and R₃ are each independently selected from —H, —OH, —F, —Cl, and—Br; R₄ is selected from —CO₂H, —P(═O)(OH)₂, —SO₃H; R₅ is selected from—COCH₃, —COCF₃, —SO₂CH₃; X is selected from —O— and —S—; Y is selectedfrom

R₆ is selected from

R₇ is selected from H, C1-C20 alkyl, C3-C20 cycloalkyl, C3-C20heterocycloalkyl; C5-C15 aryl, and C2-C15 heteroaryl; R₈ is selectedfrom C3-C20 heterocycloalkyl, C5-C15 aryl, and C2-C15 heteroaryl; Ecomprises an Fc domain monomer or an Fc domain (e.g., an Fc domainmonomer or an Fc domain, each Fc domain monomer having, independently,the sequence of any one of SEQ ID NOs: 1-68); L in each A₁-L-A₂ is alinker covalently attached to a sulfur atom of a hinge cysteine in E andto each of A₁ and A₂; T is an integer from 1 to 20 (e.g., 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), and thesquiggly line connected to the E indicates that each A₁-L-A₂ iscovalently (e.g., by way of a covalent bond or a linker) attached to asulfur atom of a hinge cysteine in E, or a pharmaceutically acceptablesalt thereof. When T is greater than 1 (e.g., T is 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), each A₁-L-A₂ may beindependently selected (e.g., independently selected from any of theA₁-L-A₂ structures described herein).

In another aspect, the invention features a conjugate described byformula (3):

wherein each A₁ and each A₂ is independently selected from any one offormulas (A-I)-(A-V):

wherein R₁ is selected from —OH, —NH₂, —NHC(═NH)NH₂, and —NHC(═NH)NHR₆;R₂ and R₃ are each independently selected from —H, —OH, —F, —Cl, and—Br; R₄ is selected from —CO₂H, —P(═O)(OH)₂, —SO₃H; R₅ is selected from—COCH₃, —COCF₃, —SO₂CH₃; X is selected from —O— and —S—; Y is selectedfrom

R₆ is selected from

R₇ is selected from H, C1-C20 alkyl, C3-C20 cycloalkyl, C3-C20heterocycloalkyl; C5-C15 aryl, and C2-C15 heteroaryl; R₈ is selectedfrom C3-C20 heterocycloalkyl, C5-C15 aryl, and C2-C15 heteroaryl; Ecomprises an Fc domain monomer or an Fc domain (e.g., an Fc domainmonomer or an Fc domain, each Fc domain monomer having, independently,the sequence of any one of SEQ ID NOs: 1-68); L in each A₁-L-A₂ is alinker covalently attached to a sulfur atom of a hinge cysteine in E andto each of A₁ and A₂; T is an integer from 1 to 20 (e.g., 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), and thesquiggly line connected to the E indicates that each A₁-L-A₂ iscovalently attached (e.g., by way of a covalent bond or a linker) to asulfur atom of a hinge cysteine in E, or a pharmaceutically acceptablesalt thereof. When T is greater than 1 (e.g., T is 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), each A₁-L-A₂ may beindependently selected (e.g., independently selected from any of theA₁-L-A₂ structures described herein).

In another aspect, the invention features a conjugate described byformula (3):

wherein each A₁ and each A₂ is independently selected from any one offormulas (A-VI)-(A-IX):

wherein R₁ is selected from —OH, —NH₂, —NHC(═NH)NH₂, and —NHC(═NH)NHR₆;R₂ and R₃ are each independently selected from —H, —OH, —F, —Cl, and—Br; R₄ is selected from —CO₂H, —P(═O)(OH)₂, —SO₃H; R₅ is selected from—COCH₃, —COCF₃, —SO₂CH₃; X is selected from —O— and —S—; Y is selectedfrom

R₆ is selected from

R₇ is selected from H, C1-C20 alkyl, C3-C20 cycloalkyl, C3-C20heterocycloalkyl; C5-C15 aryl, and C2-C15 heteroaryl; R₈ is selectedfrom C3-C20 heterocycloalkyl, C5-C15 aryl, and C2-C15 heteroaryl; Ecomprises an Fc domain monomer or an Fc domain (e.g., an Fc domainmonomer or an Fc domain, each Fc domain monomer having, independently,the sequence of any one of SEQ ID NOs: 1-68); L in each A₁-L-A₂ is alinker covalently attached to a sulfur atom of a hinge cysteine in E andto each of A₁ and A₂; T is an integer from 1 to 20 (e.g., 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), and thesquiggly line connected to the E indicates that each A₁-L-A₂ iscovalently attached (e.g., by way of a covalent bond or a linker) to asulfur atom of a hinge cysteine in E, or a pharmaceutically acceptablesalt thereof. When T is greater than 1 (e.g., T is 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), each A₁-L-A₂ may beindependently selected (e.g., independently selected from any of theA₁-L-A₂ structures described herein).

In some embodiments, each E includes an Fc domain monomer having thesequence of any one of SEQ ID NOs: 1-68.

In some embodiments, each E comprises the sequence

(SEQ ID NO: 10) MVRSDKTHTCPPCPPC*KC*PAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKHHHHHH.

In some embodiments, each E comprises the sequence

(SEQ ID NO: 11) MVRSDKTHTCPPCPPC*KC*PAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

In some embodiments, at least one of the pair of sulfur atoms is thesulfur atom corresponding to (e.g., the sulfur atom of) a hinge cysteineof SEQ ID NO: 10 or SEQ ID NO: 11, i.e., Cys10, Cys13, Cys16, or Cys18of SEQ ID NO: 10 or SEQ ID NO: 11. In some embodiments, the pair ofsulfur atoms are the sulfur atoms corresponding to (e.g., the sulfuratoms of) Cys10 and Cys13 in SEQ ID NO: 10 or SEQ ID NO: 11, Cys10 andCys16 in SEQ ID NO: 10 or SEQ ID NO: 11, Cys 30 and Cys18 in SEQ ID NO:10 or SEQ ID NO: 11, Cys13 and Cys 36 in SEQ ID NO: 10 or SEQ ID NO: 11,Cys13 and Cys 38 in SEQ ID NO: 10 or SEQ ID NO: 11, and/or Cys 36 andCys 38 in SEQ ID NO: 10 or SEQ ID NO: 11.

In some embodiments, when T is 2, the pair of sulfur atoms are thesulfur atoms corresponding to (e.g., the sulfur atoms of) Cys10 andCys13 in SEQ ID NO: 10 or SEQ ID NO: 11 and Cys 36 and Cys 38 in SEQ IDNO: 10 or SEQ ID NO: 11.

In some embodiments, the pair of sulfur atoms include one sulfur atom ofa cysteine from each E, i.e., L-A along with the sulfur atoms to whichit is attached forms a bridge between two Fc domains (e.g., two Fcdomains comprising the sequence of SEQ ID NO: 10 or SEQ ID NO: 11). Insome embodiments, the pair of sulfur atoms are the sulfur atomcorresponding to (e.g., the sulfur atom of) Cys10 of SEQ ID NO: 10 orSEQ ID NO: 11 from one E and the sulfur atom corresponding to (e.g., thesulfur atom of) Cys10 of SEQ ID NO: 10 or SEQ ID NO: 11 from another E.In some embodiments, the pair of sulfur atoms are the sulfur atomcorresponding to (e.g., the sulfur atom of) Cys13 of SEQ ID NO: 10 orSEQ ID NO: 11 from one E and the sulfur atom corresponding to (e.g., thesulfur atom of) Cys13 of SEQ ID NO: 10 or SEQ ID NO: 11 from another E.In some embodiments, the pair of sulfur atoms are the sulfur atomcorresponding to (e.g., the sulfur atom of) Cys16 of SEQ ID NO: 10 orSEQ ID NO: 11 from one E and the sulfur atom corresponding to (e.g., thesulfur atom of) Cys16 of SEQ ID NO: 10 or SEQ ID NO: 11 from another E.In some embodiments, the pair of sulfur atoms are the sulfur atomcorresponding to (e.g., the sulfur atom of) Cys18 of SEQ ID NO: 10 orSEQ ID NO: 11 from one E and the sulfur atom corresponding to (e.g., thesulfur atom of) Cys18 of SEQ ID NO: 10 or SEQ ID NO: 11 from another E.

In some embodiments, when T is 2, the pairs of sulfur atoms are thesulfur atom corresponding to (e.g., the sulfur atom of) Cys10 of SEQ IDNO: 10 or SEQ ID NO: 11 from one E and the sulfur atom corresponding to(e.g., the sulfur atom of) Cys10 of SEQ ID NO: 10 or SEQ ID NO: 11 fromanother E and the sulfur atom corresponding to (e.g., the sulfur atomof) Cys13 of SEQ ID NO: 10 or SEQ ID NO: 11 from one E and the sulfuratom corresponding to (e.g., the sulfur atom of) Cys13 of SEQ ID NO: 10or SEQ ID NO: 11 from another E. In some embodiments, when T is 2, thepairs of sulfur atoms are the sulfur atom corresponding to (e.g., thesulfur atom of) Cys10 of SEQ ID NO: 10 or SEQ ID NO: 11 from one E andthe sulfur atom corresponding to (e.g., the sulfur atom of) Cys10 of SEQID NO: 10 or SEQ ID NO: 11 from another E and the sulfur atomcorresponding to (e.g., the sulfur atom of) Cys16 of SEQ ID NO: 10 orSEQ ID NO: 11 from one E and the sulfur atom corresponding to (e.g., thesulfur atom of) Cys16 of SEQ ID NO: 10 or SEQ ID NO: 11 from another E.In some embodiments, when T is 2, the pairs of sulfur atoms are thesulfur atom corresponding to (e.g., the sulfur atom of) Cys10 of SEQ IDNO: 10 or SEQ ID NO: 11 from one E and the sulfur atom corresponding to(e.g., the sulfur atom of) Cys10 of SEQ ID NO: 10 or SEQ ID NO: 11 fromanother E and the sulfur atom corresponding to (e.g., the sulfur atomof) Cys18 of SEQ ID NO: 10 or SEQ ID NO: 11 from one E and the sulfuratom corresponding to (e.g., the sulfur atom of) Cys18 of SEQ ID NO: 10or SEQ ID NO: 11 from another E.

In some embodiments, when T is 2, the pairs of sulfur atoms are thesulfur atom corresponding to (e.g., the sulfur atom of) Cys13 of SEQ IDNO: 10 or SEQ ID NO: 11 from one E and the sulfur atom corresponding to(e.g., the sulfur atom of) Cys13 of SEQ ID NO: 10 or SEQ ID NO: 11 fromanother E and the sulfur atom corresponding to (e.g., the sulfur atomof) Cys16 of SEQ ID NO: 10 or SEQ ID NO: 11 from one E and the sulfuratom corresponding to (e.g., the sulfur atom of) Cys16 of SEQ ID NO: 10or SEQ ID NO: 11 from another E. In some embodiments, when T is 2, thepairs of sulfur atoms are the sulfur atom corresponding to (e.g., thesulfur atom of) Cys13 of SEQ ID NO: 10 or SEQ ID NO: 11 from one E andthe sulfur atom corresponding to (e.g., the sulfur atom of) Cys13 of SEQID NO: 10 or SEQ ID NO: 11 from another E and the sulfur atomcorresponding to (e.g., the sulfur atom of) Cys18 of SEQ ID NO: 10 orSEQ ID NO: 11 from one E and the sulfur atom corresponding to (e.g., thesulfur atom of) Cys18 of SEQ ID NO: 10 or SEQ ID NO: 11 from another E.

In some embodiments, when T is 2, the pairs of sulfur atoms are thesulfur atom corresponding to (e.g., the sulfur atom of) Cys16 of SEQ IDNO: 10 or SEQ ID NO: 11 from one E and the sulfur atom corresponding to(e.g., the sulfur atom of) Cys16 of SEQ ID NO: 10 or SEQ ID NO: 11 fromanother E and the sulfur atom corresponding to (e.g., the sulfur atomof) Cys18 of SEQ ID NO: 10 or SEQ ID NO: 11 from one E and the sulfuratom corresponding to (e.g., the sulfur atom of) Cys18 of SEQ ID NO: 10or SEQ ID NO: 11 from another E.

In some embodiments, when T is 3, the pairs of sulfur atoms are thesulfur atom corresponding to (e.g., the sulfur atom of) Cys10 of SEQ IDNO: 10 or SEQ ID NO: 11 from one E and the sulfur atom corresponding to(e.g., the sulfur atom of) Cys10 of SEQ ID NO: 10 or SEQ ID NO: 11 fromanother E; the sulfur atom corresponding to (e.g., the sulfur atom of)Cys13 of SEQ ID NO: 10 or SEQ ID NO: 11 from one E and the sulfur atomcorresponding to (e.g., the sulfur atom of) Cys13 of SEQ ID NO: 10 orSEQ ID NO: 11 from another E; and the sulfur atom corresponding to(e.g., the sulfur atom of) Cys16 of SEQ ID NO: 10 or SEQ ID NO: 11 fromone E and the sulfur atom corresponding to (e.g., the sulfur atom of)Cys16 of SEQ ID NO: 10 or SEQ ID NO: 11 from another E. In someembodiments, when T is 3, the pairs of sulfur atoms are the sulfur atomcorresponding to (e.g., the sulfur atom of) Cys10 of SEQ ID NO: 10 orSEQ ID NO: 11 from one E and the sulfur atom corresponding to (e.g., thesulfur atom of) Cys10 of SEQ ID NO: 10 or SEQ ID NO: 11 from another E;the sulfur atom corresponding to (e.g., the sulfur atom of) Cys13 of SEQID NO: 10 or SEQ ID NO: 11 from one E and the sulfur atom correspondingto (e.g., the sulfur atom of) Cys13 of SEQ ID NO: 10 or SEQ ID NO: 11from another E; and the sulfur atom corresponding to (e.g., the sulfuratom of) Cys18 of SEQ ID NO: 10 or SEQ ID NO: 11 from one E and thesulfur atom corresponding to (e.g., the sulfur atom of) Cys18 of SEQ IDNO: 10 or SEQ ID NO: 11 from another E. In some embodiments, when T is3, the pairs of sulfur atoms are the sulfur atom corresponding to (e.g.,the sulfur atom of) Cys10 of SEQ ID NO: 10 or SEQ ID NO: 11 from one Eand the sulfur atom corresponding to (e.g., the sulfur atom of) Cys10 ofSEQ ID NO: 10 or SEQ ID NO: 11 from another E; the sulfur atomcorresponding to (e.g., the sulfur atom of) Cys18 of SEQ ID NO: 10 orSEQ ID NO: 11 from one E and the sulfur atom corresponding to (e.g., thesulfur atom of) Cys18 of SEQ ID NO: 10 or SEQ ID NO: 11 from another E;and the sulfur atom corresponding to (e.g., the sulfur atom of) Cys16 ofSEQ ID NO: 10 or SEQ ID NO: 11 from one E and the sulfur atomcorresponding to (e.g., the sulfur atom of) Cys16 of SEQ ID NO: 10 orSEQ ID NO: 11 from another E. In some embodiments, when T is 3, thepairs of sulfur atoms are the sulfur atom corresponding to (e.g., thesulfur atom of) Cys13 of SEQ ID NO: 10 or SEQ ID NO: 11 from one E andthe sulfur atom corresponding to (e.g., the sulfur atom of) Cys13 of SEQID NO: 10 or SEQ ID NO: 11 from another E; the sulfur atom correspondingto (e.g., the sulfur atom of) Cys18 of SEQ ID NO: 10 or SEQ ID NO: 11from one E and the sulfur atom corresponding to (e.g., the sulfur atomof) Cys18 of SEQ ID NO: 10 or SEQ ID NO: 11 from another E; and thesulfur atom corresponding to (e.g., the sulfur atom of) Cys16 of SEQ IDNO: 10 or SEQ ID NO: 11 from one E and the sulfur atom corresponding to(e.g., the sulfur atom of) Cys16 of SEQ ID NO: 10 or SEQ ID NO: 11 fromanother E.

In some embodiments, when T is 3, the pairs of sulfur atoms are thesulfur atom corresponding to (e.g., the sulfur atom of) Cys10 of SEQ IDNO: 10 or SEQ ID NO: 11 from one E and the sulfur atom corresponding to(e.g., the sulfur atom of) Cys10 of SEQ ID NO: 10 or SEQ ID NO: 11 fromanother E; the sulfur atom corresponding to (e.g., the sulfur atom of)Cys13 of SEQ ID NO: 10 or SEQ ID NO: 11 from one E and the sulfur atomcorresponding to (e.g., the sulfur atom of) Cys13 of SEQ ID NO: 10 orSEQ ID NO: 11 from another E; the sulfur atom corresponding to (e.g.,the sulfur atom of) Cys16 of SEQ ID NO: 10 or SEQ ID NO: 11 from one Eand the sulfur atom corresponding to (e.g., the sulfur atom of) Cys16 ofSEQ ID NO: 10 or SEQ ID NO: 11 from another E; and the sulfur atomcorresponding to (e.g., the sulfur atom of) Cys18 of SEQ ID NO: 10 orSEQ ID NO: 11 from one E and the sulfur atom corresponding to (e.g., thesulfur atom of) Cys18 of SEQ ID NO: 10 or SEQ ID NO: 11 from another E.

In some embodiments, the conjugate has the structure:

wherein each of a, b, c, and d is, independently, 0 or 1 and whereinwhen a, b, c, or d is 0, the two sulfur atoms form a disulfide bond.

In some embodiments, a is 1 and b, c, and d are 0. In some embodiments,a and b are 1 and c and d are 0. In some embodiments, a and c are 1 andb and d are 0. In some embodiments, a and d are 1 and b and c are 0. Insome embodiments, a, b, and c are 1 and d is 0. In some embodiments, a,b, and d are 1 and c is 0. In some embodiments, a, c, and d are 1 and bis 0. In some embodiments, b and c are 1 and a and d are 0. In someembodiments, b and d are 1 and a and c are 0. In some embodiments, b, c,and d are 1 and a is 0. In some embodiments, c and d are 1 and a and bare 0. In some embodiments, a, b, c, and d are 1.

In some embodiments, each E comprises the sequence

(SEQ ID NO: 4) MVRSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

In some embodiments, each E comprises the sequence

(SEQ ID NO: 33) MVRSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

In some embodiments, at least one of the pair of sulfur atoms is thesulfur atom corresponding to (e.g., the sulfur atom of) a hinge cysteineof SEQ ID NO: 4 or SEQ ID NO: 33, i.e., Cys10 and/or Cys13. In someembodiments, the pair of sulfur atoms are the sulfur atoms correspondingto (e.g., the sulfur atoms of) Cys10 and Cys13 in SEQ ID NO: 4 or SEQ IDNO: 33.

In some embodiments, the pair of sulfur atoms include one sulfur atom ofa cysteine from each E, i.e., L-A along with the sulfur atoms to whichit is attached forms a bridge between two Fc domains (e.g., two Fcdomains comprising the sequence of SEQ ID NO: 4 or SEQ ID NO: 33). Insome embodiments, the pair of sulfur atoms are the sulfur atomcorresponding to (e.g., the sulfur atom of) Cys10 of SEQ ID NO: 4 or SEQID NO: 33 from one E and the sulfur atom corresponding to (e.g., thesulfur atom of) Cys10 of SEQ ID NO: 4 or SEQ ID NO: 33 from another E.In some embodiments, the pair of sulfur atoms are the sulfur atomcorresponding to (e.g., the sulfur atom of) Cys13 of SEQ ID NO: 4 or SEQID NO: 33 from one E and the sulfur atom corresponding to (e.g., thesulfur atom of) Cys13 of SEQ ID NO: 4 or SEQ ID NO: 33 from another E.In some embodiments, when T is 2, the pairs of sulfur atoms are thesulfur atom corresponding to (e.g., the sulfur atom of) Cys10 of SEQ IDNO: 4 or SEQ ID NO: 33 from one E and the sulfur atom corresponding to(e.g., the sulfur atom of) Cys10 of SEQ ID NO: 4 or SEQ ID NO: 33 fromanother E and the sulfur atom corresponding to (e.g., the sulfur atomof) Cys13 of SEQ ID NO: 4 or SEQ ID NO: 33 from one E and the sulfuratom corresponding to (e.g., the sulfur atom of) Cys13 of SEQ ID NO: 4or SEQ ID NO: 33 from another E.

In some embodiments, the conjugate has the structure:

wherein each of a and b is, independently, 0 or 1 and wherein when a orb is 0, the two sulfur atoms form a disulfide bond. In some embodiments,a is 1 and b is 0. In some embodiments, a is 0 and b is 1. In someembodiments, a and b are 1.

In some embodiments, each E comprises the sequence

(SEQ ID NO: 8) MVRSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKHHHHHH.

In some embodiments, at least one of the pair of sulfur atoms is thesulfur atom corresponding to (e.g., the sulfur atom of) a hinge cysteineof SEQ ID NO: 8, i.e., Cys10 and/or Cys13. In some embodiments, the pairof sulfur atoms are the sulfur atoms corresponding to (e.g., the sulfuratoms of) Cys10 and Cys13 in SEQ ID NO: 8.

In some embodiments, the pair of sulfur atoms include one sulfur atom ofa cysteine from each E, i.e., L-A along with the sulfur atoms to whichit is attached forms a bridge between two Fc domains (e.g., two Fcdomains comprising the sequence of SEQ ID NO: 8). In some embodiments,the pair of sulfur atoms are the sulfur atom corresponding to (e.g., thesulfur atom of) Cys10 of SEQ ID NO: 8 from one E and the sulfur atomcorresponding to (e.g., the sulfur atom of) Cys10 of SEQ ID NO: 8 fromanother E. In some embodiments, the pair of sulfur atoms are the sulfuratom corresponding to (e.g., the sulfur atom of) Cys13 of SEQ ID NO: 8from one E and the sulfur atom corresponding to (e.g., the sulfur atomof) Cys13 of SEQ ID NO: 8 from another E. In some embodiments, when T is2, the pairs of sulfur atoms are the sulfur atom corresponding to (e.g.,the sulfur atom of) Cys10 of SEQ ID NO: 8 from one E and the sulfur atomcorresponding to (e.g., the sulfur atom of) Cys10 of SEQ ID NO: 8 fromanother E and the sulfur atom corresponding to (e.g., the sulfur atomof) Cys13 of SEQ ID NO: 8 from one E and the sulfur atom correspondingto (e.g., the sulfur atom of) Cys13 of SEQ ID NO: 8 from another E.

In some embodiments, the conjugate has the structure:

wherein each of a and b is, independently, 0 or 1 and wherein when s orb is 0, the two sulfur atoms form a disulfide bond. In some embodiments,a is 1 and b is 0. In some embodiments, a is 0 and b is 1. In someembodiments, a and b are 1.

In another aspect, the invention features a conjugate described byformula (4):

wherein each A₁ is independently selected from any one of formulas(A-I)-(A-XII):

wherein R₁ is selected from —OH, —NH₂, —NHC(═NH)NH₂, and —NHC(═NH)NHR₆;R₂ and R₃ are each independently selected from —H, —OH, —F, —Cl, and—Br; R₄ is selected from —CO₂H, —P(═O)(OH)₂, —SO₃H; R₅ is selected from—COCH₃, —COCF₃, —SO₂CH₃; X is selected from —O— and —S—; Y is selectedfrom

R₆ is selected from

R₇ is selected from H, C1-C20 alkyl, C3-C20 cycloalkyl, C3-C20heterocycloalkyl; C5-C15 aryl, and C2-C15 heteroaryl; R₈ is selectedfrom C3-C20 heterocycloalkyl, C5-C15 aryl, and C2-C15 heteroaryl; Ecomprises an Fc domain monomer or an Fc domain (e.g., an Fc domainmonomer or an Fc domain, each Fc domain monomer having, independently,the sequence of any one of SEQ ID NOs: 1-68); L in each L-A₁ is a linkercovalently attached to a sulfur atom of a hinge cysteine in E and to A₁;T is an integer from 1 to 20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, or 20); and the squiggly line connectedto E indicates that each L-A₁ is covalently attached (e.g., by way of acovalent bond or a linker) to the sulfur atom of the hinge cysteine inE, or a pharmaceutically acceptable salt thereof. When T is greater than1 (e.g., T is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, or 20), each A₁ may be independently selected from any one offormulas (A-I)-(A-XII).

In another aspect, the invention features a conjugate described byformula (4):

wherein each A₁ is independently selected from any one of formulas(A-I)-(A-V):

wherein R₁ is selected from —OH, —NH₂, —NHC(═NH)NH₂, and —NHC(═NH)NHR₆;R₂ and R₃ are each independently selected from —H, —OH, —F, —Cl, and—Br; R₄ is selected from —CO₂H, —P(═O)(OH)₂, —SO₃H; R₅ is selected from—COCH₃, —COCF₃, —SO₂CH₃; X is selected from —O— and —S—; Y is selectedfrom

R₆ is selected from

R₇ is selected from H, C1-C20 alkyl, C3-C20 cycloalkyl, C3-C20heterocycloalkyl; C5-C15 aryl, and C2-C15 heteroaryl; R₈ is selectedfrom C3-C20 heterocycloalkyl, C5-C15 aryl, and C2-C15 heteroaryl; Ecomprises an Fc domain monomer or an Fc domain (e.g., an Fc domainmonomer or an Fc domain, each Fc domain monomer having, independently,the sequence of any one of SEQ ID NOs: 1-68); L in each L-A₁ is a linkercovalently attached to a sulfur atom of a hinge cysteine in E and to A₁;T is an integer from 1 to 20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, or 20); and the squiggly line connectedto E indicates that each L-A₁ is covalently attached (e.g., by way of acovalent bond or a linker) to the sulfur atom of the hinge cysteine inE, or a pharmaceutically acceptable salt thereof. When T is greater than1 (e.g., T is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, or 20), each A₁ may be independently selected from any one offormulas (A-I)-(A-V).

In another aspect, the invention features a conjugate described byformula (4):

wherein each A₁ is independently selected from any one of formulas(A-VI)-(A-IX):

wherein R₁ is selected from —OH, —NH₂, —NHC(═NH)NH₂, and —NHC(═NH)NHR₆;R₂ and R₃ are each independently selected from —H, —OH, —F, —Cl, and—Br; R₄ is selected from —CO₂H, —P(═O)(OH)₂, —SO₃H; R₅ is selected from—COCH₃, —COCF₃, —SO₂CH₃; X is selected from —O— and —S—; Y is selectedfrom

R₆ is selected from

R₇ is selected from H, C1-C20 alkyl, C3-C20 cycloalkyl, C3-C20heterocycloalkyl; C5-C15 aryl, and C2-C15 heteroaryl; R₈ is selectedfrom C3-C20 heterocycloalkyl, C5-C15 aryl, and C2-C15 heteroaryl; Ecomprises an Fc domain monomer or an Fc domain (e.g., an Fc domainmonomer or an Fc domain, each Fc domain monomer having, independently,the sequence of any one of SEQ ID NOs: 1-68); L in each L-A₁ is a linkercovalently attached to a sulfur atom of a hinge cysteine in E and to A₁;T is an integer from 1 to 20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, or 20); and the squiggly line connectedto E indicates that each L-A₁ is covalently attached (e.g., by way of acovalent bond or a linker) to the sulfur atom of the hinge cysteine inE, or a pharmaceutically acceptable salt thereof. When T is greater than1 (e.g., T is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, or 20), each A₁ may be independently selected from any one offormulas (A-VI)-(A-IX).

In some embodiments, each E includes an Fc domain monomer having thesequence of any one of SEQ ID NOs: 1-68.

In some embodiments, each E comprises the sequence

(SEQ ID NO: 4) MVRSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

In some embodiments, each E comprises the sequence

(SEQ ID NO: 33) MVRSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

In some embodiments, at least one of the sulfur atoms is the sulfur atomcorresponding to (e.g., the sulfur atom of) a hinge cysteine of SEQ IDNO: 4 or SEQ ID NO: 33, i.e., Cys10 and/or Cys13. In some embodiments,the sulfur atoms is the sulfur atom corresponding to (e.g., the sulfuratom of) Cys10 in SEQ ID NO: 4 or SEQ ID NO: 33. In some embodiments,the sulfur atom corresponding to (e.g., the sulfur atom of) Cys10 in SEQID NO: 4 or SEQ ID NO: 33.

In some embodiments, the conjugate has the structure:

wherein each of a and b is, independently, 0 or 1 and wherein when a orb is 0, the sulfur atoms is a thiol. In some embodiments, a is 1 and bis 0. In some embodiments, a is 0 and b is 1. In some embodiments, a andb are 1.

In another aspect, the invention features a population of conjugates,wherein the average value of T is 1 to 20 (e.g., the average value of Tis 1 to 2, 1 to 3, 1 to 4, 1 to 5, 5 to 10, 10 to 15, or 15 to 20). Insome embodiments, the average value of T is 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.

In another aspect, the invention features a conjugate described byformula (3):

wherein each A₁ and each A₂ is independently selected from any one offormulas (A-I)-(A-XII):

wherein R₁ is selected from —OH, —NH₂, —NHC(═NH)NH₂, and —NHC(═NH)NHR₆;R₂ and R₃ are each independently selected from —H, —OH, —F, —Cl, and—Br; R₄ is selected from —CO₂H, —P(═O)(OH)₂, —SO₃H; R₅ is selected from—COCH₃, —COCF₃, —SO₂CH₃; X is selected from —O— and —S—; Y is selectedfrom

R₆ is selected from

R₇ is selected from H, C1-C20 alkyl, C3-C20 cycloalkyl, C3-C20heterocycloalkyl; C5-C15 aryl, and C2-C15 heteroaryl; R₈ is selectedfrom C3-C20 heterocycloalkyl, C5-C15 aryl, and C2-C15 heteroaryl; Ecomprises an Fc domain monomer or an Fc domain (e.g., an Fc domainmonomer or an Fc domain, each Fc domain monomer having, independently,the sequence of any one of SEQ ID NOs: 1-68); L in each A₁-L-A₂ is alinker covalently attached to a nitrogen atom of a surface exposedlysine in E and to each of A₁ and A₂; T is an integer from 1 to 20(e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, or 20), and the squiggly line connected to the E indicates that eachA₁-L-A₂ is covalently attached (e.g., by way of a covalent bond or alinker) to the nitrogen atom of a surface exposed lysine in E, or apharmaceutically acceptable salt thereof. When T is greater than 1(e.g., T is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, or 20), each A₁-L-A₂ may be independently selected (e.g.,independently selected from any of the A₁-L-A₂ structures describedherein).

In another aspect, the invention features a conjugate described byformula (3):

wherein each A₁ and each A₂ is independently selected from any one offormulas (A-I)-(A-V):

wherein R₁ is selected from —OH, —NH₂, —NHC(═NH)NH₂, and —NHC(═NH)NHR₆;R₂ and R₃ are each independently selected from —H, —OH, —F, —Cl, and—Br; R₄ is selected from —CO₂H, —P(═O)(OH)₂, —SO₃H; R₅ is selected from—COCH₃, —COCF₃, —SO₂CH₃; X is selected from —O— and —S—; Y is selectedfrom

R₆ is selected from

R₇ is selected from H, C1-C20 alkyl, C3-C20 cycloalkyl, C3-C20heterocycloalkyl; C5-C15 aryl, and C2-C15 heteroaryl; R₈ is selectedfrom C3-C20 heterocycloalkyl, C5-C15 aryl, and C2-C15 heteroaryl; Ecomprises an Fc domain monomer or an Fc domain (e.g., an Fc domainmonomer or an Fc domain, each Fc domain monomer having, independently,the sequence of any one of SEQ ID NOs: 1-68); L in each A₁-L-A₂ is alinker covalently attached to a nitrogen atom of a surface exposedlysine in E and to each of A₁ and A₂; T is an integer from 1 to 20(e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, or 20), and the squiggly line connected to the E indicates that eachA₁-L-A₂ is covalently attached (e.g., by way of a covalent bond or alinker) to the nitrogen atom of a surface exposed lysine in E, or apharmaceutically acceptable salt thereof. When T is greater than 1(e.g., T is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, or 20), each A₁-L-A₂ may be independently selected (e.g.,independently selected from any of the A₁-L-A₂ structures describedherein).

In another aspect, the invention features a conjugate described byformula (3):

wherein each A₁ and each A₂ is independently selected from any one offormulas (A-VI)-(A-IX):

wherein R₁ is selected from —OH, —NH₂, —NHC(═NH)NH₂, and —NHC(═NH)NHR₆;R₂ and R₃ are each independently selected from —H, —OH, —F, —Cl, and—Br; R₄ is selected from —CO₂H, —P(═O)(OH)₂, —SO₃H; R₅ is selected from—COCH₃, —COCF₃, —SO₂CH₃; X is selected from —O— and —S—; Y is selectedfrom

R₆ is selected from

R₇ is selected from H, C1-C20 alkyl, C3-C20 cycloalkyl, C3-C20heterocycloalkyl; C5-C15 aryl, and C2-C15 heteroaryl; R₈ is selectedfrom C3-C20 heterocycloalkyl, C5-C15 aryl, and C2-C15 heteroaryl; Ecomprises an Fc domain monomer or an Fc domain (e.g., an Fc domainmonomer or an Fc domain, each Fc domain monomer having, independently,the sequence of any one of SEQ ID NOs: 1-68); L in each A₁-L-A₂ is alinker covalently attached to a nitrogen atom of a surface exposedlysine in E and to each of A₁ and A₂; T is an integer from 1 to 20(e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, or 20), and the squiggly line connected to the E indicates that eachA₁-L-A₂ is covalently attached (e.g., by way of a covalent bond or alinker) to the nitrogen atom of a surface exposed lysine in E, or apharmaceutically acceptable salt thereof. When T is greater than 1(e.g., T is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, or 20), each A₁-L-A₂ may be independently selected (e.g.,independently selected from any of the A₁-L-A₂ structures describedherein).

In another aspect, the invention features, a conjugate described byformula (4):

wherein each A₁ is independently selected from any one of formulas(A-I)-(A-XII):

wherein R₁ is selected from —OH, —NH₂, —NHC(═NH)NH₂, and —NHC(═NH)NHR₆;R₂ and R₃ are each independently selected from —H, —OH, —F, —Cl, and—Br; R₄ is selected from —CO₂H, —P(═O)(OH)₂, —SO₃H; R₅ is selected from—COCH₃, —COCF₃, —SO₂CH₃; X is selected from —O— and —S—; Y is selectedfrom

R₆ is selected from

R₇ is selected from H, C1-C20 alkyl, C3-C20 cycloalkyl, C3-C20heterocycloalkyl; C5-C15 aryl, and C2-C15 heteroaryl; R₈ is selectedfrom C3-C20 heterocycloalkyl, C5-C15 aryl, and C2-C15 heteroaryl; Ecomprises an Fc domain monomer or an Fc domain (e.g., an Fc domainmonomer or an Fc domain, each Fc domain monomer having, independently,the sequence of any one of SEQ ID NOs: 1-68); L in each L-A₁ is a linkercovalently attached to a nitrogen atom of a surface exposed lysine in Eand to A₁; T is an integer from 1 to 20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), the squiggly lineconnected to E indicates that each L-A₁ is covalently attached (e.g., byway of a covalent bond or a linker) to the nitrogen atom of a surfaceexposed lysine in E, or a pharmaceutically acceptable salt thereof. WhenT is greater than 1 (e.g., T is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, or 20), each A may be independently selectedfrom any one of formulas (A-I)-(A-XII).

In another aspect, the invention features, a conjugate described byformula (4):

wherein each A₁ is independently selected from any one of formulas(A-I)-(A-V):

wherein R₁ is selected from —OH, —NH₂, —NHC(═NH)NH₂, and —NHC(═NH)NHR₆;R₂ and R₃ are each independently selected from —H, —OH, —F, —Cl, and—Br; R₄ is selected from —CO₂H, —P(═O)(OH)₂, —SO₃H; R₅ is selected from—COCH₃, —COCF₃, —SO₂CH₃; X is selected from —O— and —S—; Y is selectedfrom

R₆ is selected from

R₇ is selected from H, C1-C20 alkyl, C3-C20 cycloalkyl, C3-C20heterocycloalkyl; C5-C15 aryl, and C2-C15 heteroaryl; R₈ is selectedfrom C3-C20 heterocycloalkyl, C5-C15 aryl, and C2-C15 heteroaryl; Ecomprises an Fc domain monomer or an Fc domain (e.g., an Fc domainmonomer or an Fc domain, each Fc domain monomer having, independently,the sequence of any one of SEQ ID NOs: 1-68); L in each L-A₁ is a linkercovalently attached to a nitrogen atom of a surface exposed lysine in Eand to A₁; T is an integer from 1 to 20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), the squiggly lineconnected to E indicates that each L-A₁ is covalently attached (e.g., byway of a covalent bond or a linker) to the nitrogen atom of a surfaceexposed lysine in E, or a pharmaceutically acceptable salt thereof. WhenT is greater than 1 (e.g., T is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, or 20), each A₁ may be independently selectedfrom any one of formulas (A-I)-(A-V).

In another aspect, the invention features, a conjugate described byformula (4):

wherein each A₁ is independently selected from any one of formulas(A-VI)-(A-IX):

wherein R₁ is selected from —OH, —NH₂, —NHC(═NH)NH₂, and —NHC(═NH)NHR₆;R₂ and R₃ are each independently selected from —H, —OH, —F, —Cl, and—Br; R₄ is selected from —CO₂H, —P(═O)(OH)₂, —SO₃H; R₅ is selected from—COCH₃, —COCF₃, —SO₂CH₃; X is selected from —O— and —S—; Y is selectedfrom

R₆ is selected from

R₇ is selected from H, C1-C20 alkyl, C3-C20 cycloalkyl, C3-C20heterocycloalkyl; C5-C15 aryl, and C2-C15 heteroaryl; R₈ is selectedfrom C3-C20 heterocycloalkyl, C5-C15 aryl, and C2-C15 heteroaryl; Ecomprises an Fc domain monomer or an Fc domain (e.g., an Fc domainmonomer or an Fc domain, each Fc domain monomer having, independently,the sequence of any one of SEQ ID NOs: 1-68); L in each L-A₁ is a linkercovalently attached to a nitrogen atom of a surface exposed lysine in Eand to A; T is an integer from 1 to 20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), the squiggly lineconnected to E indicates that each L-A₁ is covalently attached (e.g., byway of a covalent bond or a linker) to the nitrogen atom of a surfaceexposed lysine in E, or a pharmaceutically acceptable salt thereof. WhenT is greater than 1 (e.g., T is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, or 20), each A₁ may be independently selectedfrom any one of formulas (A-VI)-(A-IX).

In another aspect, the invention features a conjugate described byformula (5):

wherein each Int is independently selected from anyone of theintermediates of Table 1; E comprises an Fc domain monomer or an Fcdomain (e.g., an Fc domain monomer or an Fc domain, each Fc domainmonomer having, independently, the sequence of any one of SEQ ID NOs:1-68); L in each L-Int is a linker covalently attached to a nitrogenatom of a surface exposed lysine in E and to Int; T is an integer from 1to 20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, or 20), the squiggly line connected to E indicates that eachL-Int is covalently attached (e.g., by way of a covalent bond or alinker) to the nitrogen atom of a surface exposed lysine or the sulfuratom of a surface exposed cysteine in E, or a pharmaceuticallyacceptable salt thereof. When T is greater than 1 (e.g., T is 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), each Intmay be independently selected from any one of the intermediates of Table1.

Intermediates of Table 1 may be conjugated to an Fc domain or Fc domainmonomer (e.g., by way of a linker) by any suitable methods known tothose of skill in the art, including any of the methods described orexemplified herein. In some embodiments, the conjugate (e.g., aconjugate described by formula (5)) comprises E, wherein E is an Fcdomain monomer or an Fc domain (e.g., an Fc domain monomer or an Fcdomain, each Fc domain monomer having, independently, the sequence ofany one of SEQ ID NOs: 1-68). In preferred embodiments, one or morenitrogen atoms of one or more surface exposed lysine residues of E orone or more sulfur atoms of one or more surface exposed cysteines in Eis covalently conjugated to a linker (e.g., a PEG₂-PEG₂₀ linker). Thelinker conjugated to E may be functionalized such that it may react toform a covalent bond with any of the Ints described herein (e.g., an Intof Table 1). In preferred embodiments, E is conjugated to a linkerfunctionalized with an azido group and the Int (e.g., an Int of Table 1)is functionalized with an alkyne group. Conjugation (e.g., by clickchemistry) of the linker-azido of E and linker-alkyne of the Int forms aconjugate of the invention, for example a conjugate described by formula(5). In yet other embodiments, E is conjugated to a linkerfunctionalized with an alkyne group and the Int (e.g., an Int ofTable 1) is functionalized with an azido group. Conjugation (e.g., byclick chemistry) of the linker-alkyne of E and linker-azido of the Intforms a conjugate of the invention, for example a conjugate described byformula (5).

TABLE 1 Intermediates Intermediate Structure Int-1

Int-2

Int-3

Int-4

Int-5

Int-6

Int-7

Int-9

Int-12

Int-13

Int-14

Int-15

Int-16

Int-17

Int-18

Int-19

Int-20

Int-21

Int-22

Int-23

Int-24

Int-25

Int-26

Int-27

Int-28

Int-30

Int-31

Int-34

Int-38

Int-39

Int-40

Int-42

Int-43

Int-44

Int-45

Int-46

Int-47

Int-48

Int-49

Int-50

Int-52

Int-53

Int-54

Int-55

Int-57

Int-58

Int-59

Int-60

Int-61

Int-62

Int-63

Int-64

Int-65

Int-67

Int-68

Int-69

Int-70

Int-71

Int-72

Int-73

Int-74

Int-75

Int-76

Int-77

Int-78

Int-79

Int-80

Int-81

Int-82

Int-83

Int-84

Int-85

Int-86

Int-87

Int-88

Int-89

Int-90

In some embodiments, each E includes an Fc domain monomer having thesequence of any one of SEQ ID NOs: 1-68.

In some embodiments, E includes the sequence

(SEQ ID NO: 4) MVRSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

In some embodiments, each E includes the sequence

(SEQ ID NO: 33) MVRSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

In some embodiments, E includes the sequence

(SEQ ID NO: 10) MVRSDKTHTCPPCPPC*KC*PAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKHHHHHH

In some embodiments, each E comprises the sequence

(SEQ ID NO: 11) MVRSDKTHTCPPCPPC*KC*PAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

In some embodiments of the previous three aspects, the nitrogen atom isthe nitrogen of a surface exposed lysine, e.g., the nitrogen atomcorresponding to (e.g., the nitrogen atom of) Lys35, Lys63, Lys77,Lys79, Lys106, Lys123, Lys129, Lys181, Lys203, Lys228, or Lys236 of SEQID NO: 10 or SEQ ID NO: 11. In some embodiments, the nitrogen atom isthe nitrogen atom corresponding to (e.g., the nitrogen atom of) Lys65,Lys79, Lys108, Lys230, and/or Lys238 of SEQ ID NO: 10 or SEQ ID NO: 11.

In some embodiments, the conjugate has the structure:

wherein each of a, b, c, d, and e is, independently, 0 or 1 and whereinwhen a, b, c, d, or e is 0, the two nitrogen atom is NH₂. In someembodiments, a is 1 and b, c, d, and e are 0. In some embodiments, b is1 and a, c, d, and e are 0. In some embodiments, c is 1 and a, b, d, ande are 0. In some embodiments, d is 1 and a, b, c, and e are 0. In someembodiments, e is 1 and a, b, c, and d are 0. In some embodiments, a andb are 1 and c, d, and e are 0. In some embodiments, a and c are 1 and b,d, and e are 0. In some embodiments, a and d are 1 and b, c, and e are0. In some embodiments, a and e are 1 and b, c, and d are 0. In someembodiments, b and c are 1 and a, d, and e are 0. In some embodiments, band d are 1 and a, c, and e are 0. In some embodiments, b and e are 1and a, c, and d are 0. In some embodiments, c and d are 1 and a, b, ande are 0. In some embodiments, c and e are 1 and a, b, and d are 0. Insome embodiments, d and e are 1 and a, b, and c are 0. In someembodiments, a, b, and c are 1 and d and e are 0. In some embodiments,a, b, and d are 1 and c and e are 0. In some embodiments, a, b, and eare 1 and c and d are 0. In some embodiments, a, c, and d are 1 and band e are 0. In some embodiments, a, c, and e are 1 and b and d are 0.In some embodiments, a, d, and e are 1 and b and c are 0. In someembodiments, b, c, and d are 1 and a and e are 0. In some embodiments,b, d, and e are 1 and a and c are 0. In some embodiments, c, d, and eare 1 and a and b are 0.

In another aspect, the invention features a population of conjugatesdescribed in any of the previous aspects, wherein the average value of Tis 1 to 20 (e.g., the average value of T is 1 to 2, 1 to 3, 1 to 4, 1 to5, 5 to 10, 10 to 15, or 15 to 20). In some embodiments, the averagevalue of T is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, or 20.

In some embodiments of the conjugates described herein, the conjugateforms a homodimer including an Fc domain.

In some embodiments of the conjugates described herein, E homodimerizeswith another E to form an Fc domain.

In another aspect, the invention features a conjugate described byformula (3):

wherein each A₁ and each A₂ is independently selected from any one offormulas (A-I)-(A-XII):

wherein R₁ is selected from —OH, —NH₂, —NHC(═NH)NH₂, and —NHC(═NH)NHR₆;R₂ and R₃ are each independently selected from —H, —OH, —F, —Cl, and—Br; R₄ is selected from —CO₂H, —P(═O)(OH)₂, —SO₃H; R₅ is selected from—COCH₃, —COCF₃, —SO₂CH₃; X is selected from —O— and —S—; Y is selectedfrom

R₆ is selected from

R₇ is selected from H, C1-C20 alkyl, C3-C20 cycloalkyl, C3-C20heterocycloalkyl; C5-C15 aryl, and C2-C15 heteroaryl; R₈ is selectedfrom C3-C20 heterocycloalkyl, C5-C15 aryl, and C2-C15 heteroaryl; Ecomprises an albumin protein (e.g., an albumin protein having thesequence of any one of SEQ ID NOs: 69-71), an albumin protein-bindingpeptide, or an Fc-binding peptide; L in each A₁-L-A₂ is a linkerindependently covalently attached to a sulfur atom of a surface exposedcysteine or a nitrogen atom of a surface exposed lysine in E and to eachof A₁ and A₂; T is an integer from 1 to 20 (e.g., 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), and the squigglyline connected to the E indicates that each A₁-L-A₂ is independentlycovalently attached (e.g., by way of a bond or a linker) to the sulfuratom of a solvent-exposed cysteine or the nitrogen atom of asolvent-exposed lysine in E, or a pharmaceutically acceptable saltthereof. When T is greater than 1 (e.g., T is 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), each A₁-L-A₂ may beindependently selected (e.g., independently selected from any of theA₁-L-A₂ structures described herein).

In another aspect, the invention features a conjugate described byformula (3):

wherein each A₁ and each A₂ is independently selected from any one offormulas (A-I)-(A-V):

wherein R₁ is selected from —OH, —NH₂, —NHC(═NH)NH₂, and —NHC(═NH)NHR₆;R₂ and R₃ are each independently selected from —H, —OH, —F, —Cl, and—Br; R₄ is selected from —CO₂H, —P(═O)(OH)₂, —SO₃H; R₅ is selected from—COCH₃, —COCF₃, —SO₂CH₃; X is selected from —O— and —S—; Y is selectedfrom

R₆ is selected from

R₇ is selected from H, C1-C20 alkyl, C3-C20 cycloalkyl, C3-C20heterocycloalkyl; C5-C15 aryl, and C2-C15 heteroaryl; R₈ is selectedfrom C3-C20 heterocycloalkyl, C5-C15 aryl, and C2-C15 heteroaryl; Ecomprises an albumin protein (e.g., an albumin protein having thesequence of any one of SEQ ID NOs: 69-71), an albumin protein-bindingpeptide, or an Fc-binding peptide; L in each A₁-L-A₂ is a linkerindependently covalently attached to a sulfur atom of a surface exposedcysteine or a nitrogen atom of a surface exposed lysine in E and to eachof A₁ and A₂; T is an integer from 1 to 20 (e.g., 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), and the squigglyline connected to the E indicates that each A₁-L-A₂ is independentlycovalently attached (e.g., by way of a bond or a linker) to the sulfuratom of a solvent-exposed cysteine or the nitrogen atom of asolvent-exposed lysine in E, or a pharmaceutically acceptable saltthereof. When T is greater than 1 (e.g., T is 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), each A₁-L-A₂ may beindependently selected (e.g., independently selected from any of theA₁-L-A₂ structures described herein).

In another aspect, the invention features a conjugate described byformula (3):

wherein each A₁ and each A₂ is independently selected from any one offormulas (A-VI)-(A-IX):

wherein R₁ is selected from —OH, —NH₂, —NHC(═NH)NH₂, and —NHC(═NH)NHR₆;R₂ and R₃ are each independently selected from —H, —OH, —F, —Cl, and—Br; R₄ is selected from —CO₂H, —P(═O)(OH)₂, —SO₃H; R₅ is selected from—COCH₃, —COCF₃, —SO₂CH₃; X is selected from —O— and —S—; Y is selectedfrom

R₆ is selected from

R₇ is selected from H, C1-C20 alkyl, C3-C20 cycloalkyl, C3-C20heterocycloalkyl; C5-C15 aryl, and C2-C15 heteroaryl; R₈ is selectedfrom C3-C20 heterocycloalkyl, C5-C15 aryl, and C2-C15 heteroaryl; Ecomprises an albumin protein (e.g., an albumin protein having thesequence of any one of SEQ ID NOs: 69-71), an albumin protein-bindingpeptide, or an Fc-binding peptide; L in each A₁-L-A₂ is a linkerindependently covalently attached to a sulfur atom of a surface exposedcysteine or a nitrogen atom of a surface exposed lysine in E and to eachof A₁ and A₂; T is an integer from 1 to 20 (e.g., 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), and the squigglyline connected to the E indicates that each A₁-L-A₂ is independentlycovalently attached (e.g., by way of a bond or a linker) to the sulfuratom of a solvent-exposed cysteine or the nitrogen atom of asolvent-exposed lysine in E, or a pharmaceutically acceptable saltthereof. When T is greater than 1 (e.g., T is 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), each A₁-L-A₂ may beindependently selected (e.g., independently selected from any of theA₁-L-A₂ structures described herein).

In another aspect, the invention features a conjugate described byformula (4):

wherein each A₁ is independently selected from any one of formulas(A-I)-(A-XII):

wherein R₁ is selected from —OH, —NH₂, —NHC(═NH)NH₂, and —NHC(═NH)NHR₆;R₂ and R₃ are each independently selected from —H, —OH, —F, —Cl, and—Br; R₄ is selected from —CO₂H, —P(═O)(OH)₂, —SO₃H, R₅ is selected from—COCH₃, —COCF₃, —SO₂CH₃; X is selected from —O— and —S—; Y is selectedfrom

R₆ is selected from

R₇ is selected from H, C1-C20 alkyl, C3-C20 cycloalkyl, C3-C20heterocycloalkyl; C5-C15 aryl, and C2-C15 heteroaryl; R₈ is selectedfrom C3-C20 heterocycloalkyl, C5-C15 aryl, and C2-C15 heteroaryl; Ecomprises an albumin protein (e.g., an albumin protein having thesequence of any one of SEQ ID NOs: 69-71), an albumin protein-bindingpeptide, or an Fc-binding peptide; L in each L-A₁ is a linkerindependently covalently attached to a sulfur atom of a surface exposedcysteine or a nitrogen atom of a surface exposed lysine in E and to A₁;T is an integer from 1 to 20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, or 20); and the squiggly line connectedto E indicates that each L-A₁ is independently covalently attached(e.g., by way of a bond or a linker) to the sulfur atom of thesolvent-exposed cysteine or the nitrogen atom of the solvent-exposedlysine in E, or a pharmaceutically acceptable salt thereof. When T isgreater than 1 (e.g., T is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, or 20), each A may be independently selected fromanyone of formulas (A-I)-(A-XII).

In another aspect, the invention features a conjugate described byformula (4):

wherein each A₁ is independently selected from any one of formulas(A-I)-(A-V):

wherein R₁ is selected from —OH, —NH₂, —NHC(═NH)NH₂, and —NHC(═NH)NHR₆;R₂ and R₃ are each independently selected from —H, —OH, —F, —Cl, and—Br; R₄ is selected from —CO₂H, —P(═O)(OH)₂, —SO₃H; R₅ is selected from—COCH₃, —COCF₃, —SO₂CH₃; X is selected from —O— and —S—; Y is selectedfrom

R₆ is selected from

R₇ is selected from H, C1-C20 alkyl, C3-C20 cycloalkyl, C3-C20heterocycloalkyl; C5-C15 aryl, and C2-C15 heteroaryl; R₈ is selectedfrom C3-C20 heterocycloalkyl, C5-C15 aryl, and C2-C15 heteroaryl; Ecomprises an albumin protein (e.g., an albumin protein having thesequence of any one of SEQ ID NOs: 69-71), an albumin protein-bindingpeptide, or an Fc-binding peptide; L in each L-A₁ is a linkerindependently covalently attached to a sulfur atom of a surface exposedcysteine or a nitrogen atom of a surface exposed lysine in E and to A₁;T is an integer from 1 to 20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, or 20); and the squiggly line connectedto E indicates that each L-A is independently covalently attached (e.g.,by way of a bond or a linker) to the sulfur atom of the solvent-exposedcysteine or the nitrogen atom of the solvent-exposed lysine in E, or apharmaceutically acceptable salt thereof. When T is greater than 1(e.g., T is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, or 20), each A₁ may be independently selected from anyone offormulas (A-I)-(A-V).

In another aspect, the invention features a conjugate described byformula (4):

wherein each A₁ is independently selected from any one of formulas(A-VI)-(A-IX):

wherein R₁ is selected from —OH, —NH₂, —NHC(═NH)NH₂, and —NHC(═NH)NHR₆;R₂ and R₃ are each independently selected from —H, —OH, —F, —Cl, and—Br; R₄ is selected from —CO₂H, —P(═O)(OH)₂, —SO₃H; R₅ is selected from—COCH₃, —COCF₃, —SO₂CH₃; X is selected from —O— and —S—; Y is selectedfrom

R₆ is selected from

R₇ is selected from H, C1-C20 alkyl, C3-C20 cycloalkyl, C3-C20heterocycloalkyl; C5-C15 aryl, and C2-C15 heteroaryl; R₈ is selectedfrom C3-C20 heterocycloalkyl, C5-C15 aryl, and C2-C15 heteroaryl; Ecomprises an albumin protein (e.g., an albumin protein having thesequence of any one of SEQ ID NOs: 69-71), an albumin protein-bindingpeptide, or an Fc-binding peptide; L in each L-A₁ is a linkerindependently covalently attached to a sulfur atom of a surface exposedcysteine or a nitrogen atom of a surface exposed lysine in E and to A₁;T is an integer from 1 to 20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, or 20); and the squiggly line connectedto E indicates that each L-A₁ is independently covalently attached(e.g., by way of a bond or a linker) to the sulfur atom of thesolvent-exposed cysteine or the nitrogen atom of the solvent-exposedlysine in E, or a pharmaceutically acceptable salt thereof. When T isgreater than 1 (e.g., T is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, or 20), each A may be independently selected fromanyone of formulas (A-VI)-(A-IX).

In another aspect, the invention features a conjugate described byformula (5):

wherein each Int is independently selected from anyone of theintermediates of Table 1; E comprises an albumin protein (e.g., analbumin protein having the sequence of any one of SEQ ID NOs: 69-71), analbumin protein-binding peptide, or an Fc-binding peptide; L in eachL-Int is a linker independently covalently attached to a sulfur atom ofa surface exposed cysteine or a nitrogen atom of a surface exposedlysine in E and to Int; T is an integer from 1 to 20 (e.g., 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20); and thesquiggly line connected to E indicates that each L-Int is independentlycovalently attached to the sulfur atom of the solvent-exposed cysteineor the nitrogen atom of the solvent-exposed lysine in E, or apharmaceutically acceptable salt thereof. When T is greater than 1(e.g., T is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, or 20), each Int may be independently selected from anyone of theintermediates of Table 1.

Intermediates of Table 1 may be conjugated to an albumin protein, analbumin protein-binding peptide, or an Fc-binding peptide (e.g., by wayof a linker) by any suitable methods known to those of skill in the art,including any of the methods described or exemplified herein. In someembodiments, the conjugate (e.g., a conjugate described by formula (5))comprises E, wherein E is an albumin protein (e.g., an albumin proteinhaving the sequence of any one of SEQ ID NOs: 69-71), an albuminprotein-binding peptide, or an Fc-binding peptide. In preferredembodiments, one or more nitrogen atoms of one or more surface exposedlysine residues of E or one or more sulfur atoms of one or more surfaceexposed cysteines in E is covalently conjugated to a linker (e.g., aPEG₂-PEG₂₀ linker). The linker conjugated to E may be functionalizedsuch that it may react to form a covalent bond with any of the Intsdescribed herein (e.g., an Int of Table 1). In preferred embodiments, Eis conjugated to a linker functionalized with an azido group and the Int(e.g., an Int of Table 1) is functionalized with an alkyne group.Conjugation (e.g., by click chemistry) of the linker-azido of E andlinker-alkyne of the Int forms a conjugate of the invention, for examplea conjugate described by formula (5). In yet other embodiments, E isconjugated to a linker functionalized with an alkyne group and the Int(e.g., an Int of Table 1) is functionalized with an azido group.Conjugation (e.g., by click chemistry) of the linker-alkyne of E andlinker-azido of the Int forms a conjugate of the invention, for examplea conjugate described by formula (5).

In some embodiments, each E includes an albumin protein having thesequence of any one of SEQ ID NOs: 69-71.

In some embodiments, T is 1 and L-A is covalently attached to the sulfuratom corresponding to Cys34 of SEQ ID NO: 69.

In another aspect, the invention features a population of conjugatesdescribed in any of the previous aspects, wherein the average value of Tis 1 to 20 (e.g., the average value of T is 1 to 2, 1 to 3, 1 to 4, 1 to5, 5 to 10, 10 to 15, or 15 to 20). In some embodiments, the averagevalue of T is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, or 20.

In another aspect, the invention features a conjugate including (i) afirst moiety, A₁; (ii) a second moiety, A₂; (iii) an Fc domain monomeror an Fc domain; and (iv) a linker covalently attached to A₁ and A₂, andto the Fc domain monomer or the Fc domain; wherein each A₁ and each A₂is independently selected from any one of formulas (A-I)-(A-XII):

wherein R₁ is selected from —OH, —NH₂, —NHC(═NH)NH₂, and —NHC(═NH)NHR₆;R₂ and R₃ are each independently selected from —H, —OH, —F, —Cl, and—Br; R₄ is selected from —CO₂H, —P(═O)(OH)₂, —SO₃H; R₅ is selected from—COCH₃, —COCF₃, —SO₂CH₃; X is selected from —O— and —S—; Y is selectedfrom

R₆ is selected from

R₇ is selected from H, C1-C20 alkyl, C3-C20 cycloalkyl, C3-C20heterocycloalkyl; C5-C15 aryl, and C2-C15 heteroaryl; R₈ is selectedfrom C3-C20 heterocycloalkyl, C5-C15 aryl, and C2-C15 heteroaryl, or apharmaceutically acceptable salt thereof.

In another aspect, the invention features a conjugate including (i) afirst moiety, A₁; (ii) a second moiety, A₂; (iii) an Fc domain monomeror an Fc domain; and (iv) a linker covalently attached to A₁ and A₂, andto the Fc domain monomer or the Fc domain; wherein each A₁ and each A₂is independently selected from any one of formulas (A-I)-(A-V):

wherein R₁ is selected from —OH, —NH₂, —NHC(═NH)NH₂, and —NHC(═NH)NHR₆;R₂ and R₃ are each independently selected from —H, —OH, —F, —Cl, and—Br; R₄ is selected from —CO₂H, —P(═O)(OH)₂, —SO₃H; R₅ is selected from—COCH₃, —COCF₃, —SO₂CH₃; X is selected from —O— and —S—; Y is selectedfrom

R₆ is selected from

R₇ is selected from H, C1-C20 alkyl, C3-C20 cycloalkyl, C3-C20heterocycloalkyl; C5-C15 aryl, and C2-C15 heteroaryl; R₈ is selectedfrom C3-C20 heterocycloalkyl, C5-C15 aryl, and C2-C15 heteroaryl, or apharmaceutically acceptable salt thereof.

In another aspect, the invention features a conjugate including (i) afirst moiety, A₁; (ii) a second moiety, A₂; (iii) an Fc domain monomeror an Fc domain; and (iv) a linker covalently attached to A₁ and A₂, andto the Fc domain monomer or the Fc domain; wherein each A₁ and each A₂is independently selected from any one of formulas (A-VI)-(A-IX):

wherein R₁ is selected from —OH, —NH₂, —NHC(═NH)NH₂, and —NHC(═NH)NHR₆;R₂ and R₃ are each independently selected from —H, —OH, —F, —Cl, and—Br; R₄ is selected from —CO₂H, —P(═O)(OH)₂, —SO₃H; R₅ is selected from—COCH₃, —COCF₃, —SO₂CH₃; X is selected from —O— and —S—; Y is selectedfrom

R₆ is selected from

R₇ is selected from H, C1-C20 alkyl, C3-C20 cycloalkyl, C3-C20heterocycloalkyl; C5-C15 aryl, and C2-C15 heteroaryl; R₈ is selectedfrom C3-C20 heterocycloalkyl, C5-C15 aryl, and C2-C15 heteroaryl, or apharmaceutically acceptable salt thereof.

In another aspect, the invention features a conjugate including (i) afirst moiety, Int; (ii) an Fc domain monomer or an Fc domain; and (iv) alinker covalently attached to Int, and to the Fc domain monomer or theFc domain; wherein each Intis independently selected from anyone of theintermediates of Table 1.

In another aspect, the invention features a conjugate including (i) afirst moiety, A₁; (ii) a second moiety, A₂; (iii) an albumin protein, analbumin protein-binding peptide, or an Fc-binding peptide; and (iv) alinker covalently attached to A₁ and A₂, and to the albumin protein, thealbumin protein-binding peptide, or the Fc-binding peptide; wherein eachA₁ and each A₂ is independently selected from any one of formulas(A-I)-(A-XII):

wherein R₁ is selected from —OH, —NH₂, —NHC(═NH)NH₂, and —NHC(═NH)NHR₆;R₂ and R₃ are each independently selected from —H, —OH, —F, —Cl, and—Br; R₄ is selected from —CO₂H, —P(═O)(OH)₂, —SO₃H; R₅ is selected from—COCH₃, —COCF₃, —SO₂CH₃; X is selected from —O— and —S—; Y is selectedfrom

R₆ is selected from

R₇ is selected from H, C1-C20 alkyl, C3-C20 cycloalkyl, C3-C20heterocycloalkyl; C5-C15 aryl, and C2-C15 heteroaryl; R₈ is selectedfrom C3-C20 heterocycloalkyl, C5-C15 aryl, and C2-C15 heteroaryl, or apharmaceutically acceptable salt thereof.

In another aspect, the invention features a conjugate including (i) afirst moiety, A₁; (ii) a second moiety, A₂; (iii) an albumin protein, analbumin protein-binding peptide, or an Fc-binding peptide; and (iv) alinker covalently attached to A₁ and A₂, and to the albumin protein, thealbumin protein-binding peptide, or the Fc-binding peptide; wherein eachA₁ and each A₂ is independently selected from any one of

wherein R₁ is selected from —OH, —NH₂, —NHC(═NH)NH₂, and —NHC(═NH)NHR₆;R₂ and R₃ are each independently selected from —H, —OH, —F, —Cl, and—Br; R₄ is selected from —CO₂H, —P(═O)(OH)₂, —SO₃H; R₅ is selected from—COCH₃, —COCF₃, —SO₂CH₃; X is selected from —O— and —S—; Y is selectedfrom

R₆ is selected from

R₇ is selected from H, C1-C20 alkyl, C3-C20 cycloalkyl, C3-C20heterocycloalkyl; C5-C15 aryl, and C2-C15 heteroaryl; R₈ is selectedfrom C3-C20 heterocycloalkyl, C5-C15 aryl, and C2-C15 heteroaryl, or apharmaceutically acceptable salt thereof.

In another aspect, the invention features a conjugate including (i) afirst moiety, A₁; (ii) a second moiety, A₂; (iii) an albumin protein, analbumin protein-binding peptide, or an Fc-binding peptide; and (iv) alinker covalently attached to A₁ and A₂, and to the albumin protein, thealbumin protein-binding peptide, or the Fc-binding peptide; wherein eachA₁ and each A₂ is independently selected from any one of formulas(A-VI)-(A-IX):

wherein R₁ is selected from —OH, —NH₂, —NHC(═NH)NH₂, and —NHC(═NH)NHR₆;R₂ and R₃ are each independently selected from —H, —OH, —F, —Cl, and—Br; R₄ is selected from —CO₂H, —P(═O)(OH)₂, —SO₃H; R₅ is selected from—COCH₃, —COCF₃, —SO₂CH₃; X is selected from —O— and —S—; Y is selectedfrom

R₆ is selected from

R₇ is selected from H, C1-C20 alkyl, C3-C20 cycloalkyl, C3-C20heterocycloalkyl; C5-C15 aryl, and C2-C15 heteroaryl; R₈ is selectedfrom C3-C20 heterocycloalkyl, C5-C15 aryl, and C2-C15 heteroaryl, or apharmaceutically acceptable salt thereof.

In another aspect, the invention features a conjugate described byformula (D-I):

wherein each A₁ and each A₂ is independently selected from any one offormulas (A-I)-(A-XII):

wherein R₁ is selected from —OH, —NH₂, —NHC(═NH)NH₂, and —NHC(═NH)NHR₆;R₂ and R₃ are each independently selected from —H, —OH, —F, —Cl, and—Br; R₄ is selected from —CO₂H, —P(═O)(OH)₂, —SO₃H; R₅ is selected from—COCH₃, —COCF₃, —SO₂CH₃; X is selected from —O— and —S—; Y is selectedfrom

R₆ is selected from

R₇ is selected from H, C1-C20 alkyl, C3-C20 cycloalkyl, C3-C20heterocycloalkyl; C5-C15 aryl, and C2-C15 heteroaryl; R₈ is selectedfrom C3-C20 heterocycloalkyl, C5-C15 aryl, and C2-C15 heteroaryl; each Ecomprises an Fc domain monomer (e.g., an Fc domain monomer having thesequence of any one of SEQ ID NOs: 1-68), an albumin protein (e.g., analbumin protein having the sequence of any one of SEQ ID NOs: 69-71), analbumin protein-binding peptide, or an Fc-binding peptide; n is 1 or 2;T is an integer from 1 to 20 (e.g., T is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, or 20); and Lis a linker covalentlyattached to each of E, A₁, and A₂, or a pharmaceutically acceptable saltthereof. When T is greater than 1 (e.g., T is 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), each A₁-L-A₂ may beindependently selected (e.g., independently selected from any of theA₁-L-A₂ structures described herein).

In another aspect, the invention features a conjugate described byformula (D-I):

wherein each A₁ and each A₂ is independently selected from any one offormulas (A-I)-(A-V):

wherein R₁ is selected from —OH, —NH₂, —NHC(═NH)NH₂, and —NHC(═NH)NHR₆;R₂ and R₃ are each independently selected from —H, —OH, —F, —Cl, and—Br; R₄ is selected from —CO₂H, —P(═O)(OH)₂, —SO₃H; R₅ is selected from—COCH₃, —COCF₃, —SO₂CH₃; X is selected from —O— and —S—; Y is selectedfrom

R₆ is selected from

R₇ is selected from H, C1-C20 alkyl, C3-C20 cycloalkyl, C3-C20heterocycloalkyl; C5-C15 aryl, and C2-C15 heteroaryl; R₈ is selectedfrom C3-C20 heterocycloalkyl, C5-C15 aryl, and C2-C15 heteroaryl; each Ecomprises an Fc domain monomer (e.g., an Fc domain monomer having thesequence of any one of SEQ ID NOs: 1-68), an albumin protein (e.g., analbumin protein having the sequence of any one of SEQ ID NOs: 69-71), analbumin protein-binding peptide, or an Fc-binding peptide; n is 1 or 2;T is an integer from 1 to 20 (e.g., T is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, or 20); and Lis a linker covalentlyattached to each of E, A₁, and A₂, or a pharmaceutically acceptable saltthereof. When T is greater than 1 (e.g., T is 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), each A₁-L-A₂ may beindependently selected (e.g., independently selected from any of theA₁-L-A₂ structures described herein).

In another aspect, the invention features a conjugate described byformula (D-I):

wherein each A₁ and each A₂ is independently selected from any one offormulas (A-VI)-(A-IX):

wherein R₁ is selected from —OH, —NH₂, —NHC(═NH)NH₂, and —NHC(═NH)NHR₆;R₂ and R₃ are each independently selected from —H, —OH, —F, —Cl, and—Br; R₄ is selected from —CO₂H, —P(═O)(OH)₂, —SO₃H; R₅ is selected from—COCH₃, —COCF₃, —SO₂CH₃; X is selected from —O— and —S—; Y is selectedfrom

R₆ is selected from

R₇ is selected from H, C1-C20 alkyl, C3-C20 cycloalkyl, C3-C20heterocycloalkyl; C5-C15 aryl, and C2-C15 heteroaryl; R₈ is selectedfrom C3-C20 heterocycloalkyl, C5-C15 aryl, and C2-C15 heteroaryl; each Ecomprises an Fc domain monomer (e.g., an Fc domain monomer having thesequence of any one of SEQ ID NOs: 1-68), an albumin protein (e.g., analbumin protein having the sequence of any one of SEQ ID NOs: 69-71), analbumin protein-binding peptide, or an Fc-binding peptide; n is 1 or 2;T is an integer from 1 to 20 (e.g., T is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, or 20); and Lis a linker covalentlyattached to each of E, A₁, and A₂, or a pharmaceutically acceptable saltthereof. When T is greater than 1 (e.g., T is 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), each A₁-L-A₂ may beindependently selected (e.g., independently selected from any of theA₁-L-A₂ structures described herein).

In some embodiments, the conjugate is described by formula (D-II):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (D-II-1):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (D-II-2):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (D-II-3):

wherein L′ is the remainder of L, and y₁ and y₂ are each independentlyan integer from 1-20 (e.g., y₁ and y₂ are each independently 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), or apharmaceutically acceptable salt thereof. In some embodiments, L′ is anitrogen atom.

In some embodiments, the conjugate has the structure selected from

In some embodiments, the conjugate is described by formula (D-II-4):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (D-II-5):

wherein L′ is the remainder of L, and y₁ and y₂ are each independentlyan integer from 1-20 (e.g., y₁ and y₂ are each independently 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), or apharmaceutically acceptable salt thereof. In some embodiments, L′ is anitrogen atom.

In some embodiments, the conjugate has the structure selected from

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate has the structure selected from

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (D-II-6):

wherein R₇ is selected from H, C1-C20 alkyl, C3-C20 cycloalkyl, C3-C20heterocycloalkyl; C5-C15 aryl, and C2-C15 heteroaryl; or apharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (D-II-7):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (D-II-8):

wherein L′ is the remainder of L, and y₁ and y₂ are each independentlyan integer from 1-20 (e.g., y₁ and y₂ are each independently 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), or apharmaceutically acceptable salt thereof. In some embodiments, L′ is anitrogen atom.

In some embodiments, the conjugate has the structure

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate has the structure

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate has the structure

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (D-II-9):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (D-II-10):

wherein L′ is the remainder of L, and y₁ and y₂ are each independentlyan integer from 1-20 (e.g., y₁ and y₂ are each independently 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), or apharmaceutically acceptable salt thereof. In some embodiments, L′ is anitrogen atom.

In some embodiments, the conjugate has the structure

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate has the structure of

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (D-III):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (D-III-1):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (D-III-2):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (D-III-3):

wherein L′ is the remainder of L, and y₁ and y₂ are each independentlyan integer from 1-20 (e.g., y₁ and y₂ are each independently 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), or apharmaceutically acceptable salt thereof. In some embodiments, L′ is anitrogen atom.

In some embodiments, the conjugate is described by formula (D-III-4):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (D-III-5):

wherein L′ is the remainder of L, and y₁ and y₂ are each independentlyan integer from 1-20 (e.g., y₁ and y₂ are each independently 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), or apharmaceutically acceptable salt thereof. In some embodiments, L′ is anitrogen atom.

In some embodiments, the conjugate is described by formula (D-III-6):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (D-III-7):

wherein L′ is the remainder of L, and y₁ and y₂ are each independentlyan integer from 1-20 (e.g., y₁ and y₂ are each independently 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), or apharmaceutically acceptable salt thereof. In some embodiments, L′ is anitrogen atom.

In some embodiments, the conjugate is described by formula (D-III-8):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (D-III-9):

wherein L′ is the remainder of L, and y₁ and y₂ are each independentlyan integer from 1-20 (e.g., y₁ and y₂ are each independently 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), or apharmaceutically acceptable salt thereof. In some embodiments, L′ is anitrogen atom.

In some embodiments, the conjugate is described by formula (D-IV):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (D-IV-1):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (D-IV-2):

wherein L′ is the remainder of L, and y₁ and y₂ are each independentlyan integer from 1-20 (e.g., y₁ and y₂ are each independently 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), or apharmaceutically acceptable salt thereof. In some embodiments, L′ is anitrogen atom. In some embodiments, the conjugate is described byformula (D-V):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (D-V-1):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (D-V-2):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (D-V-3):

wherein L′ is the remainder of L, and y₁ and y₂ are each independentlyan integer from 1-20 (e.g., y₁ and y₂ are each independently 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), or apharmaceutically acceptable salt thereof. In some embodiments, L′ is anitrogen atom. In some embodiments, y₁ and y₂ are each 1, y₁ and y₂ areeach 2, or y₁ and y₂ are each 3.

In some embodiments, the conjugate is described by formula (D-V-4):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (D-V-5):

wherein L′ is the remainder of L, and y₁ and y₂ are each independentlyan integer from 1-20 (e.g., y₁ and y₂ are each independently 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), or apharmaceutically acceptable salt thereof. In some embodiments, L′ is anitrogen atom. In some embodiments, y₁ and y₂ are each 1, y₁ and y₂ areeach 2, or y₁ and y₂ are each 3.

In some embodiments, the conjugate is described by formula (D-V-6):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (D-V-7):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (D-V-8):

wherein L′ is the remainder of L, and y₁ and y₂ are each independentlyan integer from 1-20 (e.g., y₁ and y₂ are each independently 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), or apharmaceutically acceptable salt thereof. In some embodiments, L′ is anitrogen atom. In some embodiments, y₁ and y₂ are each 1, y₁ and y₂ areeach 2, or y₁ and y₂ are each 3.

In some embodiments, the conjugate is described by formula (D-V-9):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (D-V-10):

wherein L′ is the remainder of L, and y₁ and y₂ are each independentlyan integer from 1-20 (e.g., y₁ and y₂ are each independently 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), or apharmaceutically acceptable salt thereof. In some embodiments, L′ is anitrogen atom. In some embodiments, y₁ and y₂ are each 1, y₁ and y₂ areeach 2, or y₁ and y₂ are each 3.

In some embodiments, the conjugate is described by formula (D-VI):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (D-VI-1):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (D-VI-2):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (D-VI-3):

wherein L′ is the remainder of L, and y₁ and y₂ are each independentlyan integer from 1-20 (e.g., y₁ and y₂ are each independently 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), or apharmaceutically acceptable salt thereof. In some embodiments, L′ is anitrogen atom. In some embodiments, y₁ and y₂ are each 1, y₁ and y₂ areeach 2, or y₁ and y₂ are each 3.

In some embodiments, the conjugate is described by formula (D-VI-4):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (D-VI-5):

wherein L′ is the remainder of L, and y₁ and y₂ are each independentlyan integer from 1-20 (e.g., y₁ and y₂ are each independently 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), or apharmaceutically acceptable salt thereof. In some embodiments, L′ is anitrogen atom. In some embodiments, y₁ and y₂ are each 1, y₁ and y₂ areeach 2, or y₁ and y₂ are each 3.

In some embodiments, the conjugate is described by formula (D-VI-6):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (D-VI-7):

wherein L′ is the remainder of L, and y₁ and y₂ are each independentlyan integer from 1-20 (e.g., y₁ and y₂ are each independently 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), or apharmaceutically acceptable salt thereof. In some embodiments, L′ is anitrogen atom. In some embodiments, y₁ and y₂ are each 1, y₁ and y₂ areeach 2, or y₁ and y₂ are each 3.

In some embodiments, the conjugate is described by formula (D-VI-8):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (D-VI-9):

wherein L′ is the remainder of L, and y₁ and y₂ are each independentlyan integer from 1-20 (e.g., y₁ and y₂ are each independently 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), or apharmaceutically acceptable salt thereof. In some embodiments, L′ is anitrogen atom. In some embodiments, y₁ and y₂ are each 1, y₁ and y₂ areeach 2, or y₁ and y₂ are each 3.

In some embodiments, the conjugate is described by formula (D-VII):

or a pharmaceutically acceptable salt thereof.

In some embodiments of any of the aspects described herein, R₁ is OH. Insome embodiments of any of the aspects described herein, R₁ is NH₂. Insome embodiments of any of the aspects described herein, R₁ is—NHC(═NH)NH₂.

In some embodiments, the conjugate is described by formula (D-VIII):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (D-VIII-1):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (D-VIII-2):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (D-VIII-3):

wherein L′ is the remainder of L, and y₁ and y₂ are each independentlyan integer from 1-20 (e.g., y₁ and y₂ are each independently 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), or apharmaceutically acceptable salt thereof. In some embodiments, L′ is anitrogen atom.

In some embodiments, the conjugate has the structure selected from

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (D-VIII-4):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (D-VIII-5):

wherein L′ is the remainder of L, and y₁ and y₂ are each independentlyan integer from 1-20 (e.g., y₁ and y₂ are each independently 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), or apharmaceutically acceptable salt thereof. In some embodiments, L′ is anitrogen atom.

In some embodiments, the conjugate has the structure selected from

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by the structure

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (D-VIII-6):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (D-VIII-7):

wherein L′ is the remainder of L, and y₁ and y₂ are each independentlyan integer from 1-20 (e.g., y₁ and y₂ are each independently 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), or apharmaceutically acceptable salt thereof. In some embodiments, L′ is anitrogen atom.

In some embodiments, the conjugate is described by formula (D-VIII-8):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (D-VIII-9):

wherein L′ is the remainder of L, and y₁ and y₂ are each independentlyan integer from 1-20 (e.g., y₁ and y₂ are each independently 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), or apharmaceutically acceptable salt thereof. In some embodiments, L′ is anitrogen atom.

In some embodiments, the conjugate is described by formula (D-VIII-10):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (D-VIII-11):

wherein L′ is the remainder of L, and y₁ and y₂ are each independentlyan integer from 1-20 (e.g., y₁ and y₂ are each independently 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), or apharmaceutically acceptable salt thereof. In some embodiments, L′ is anitrogen atom. In some embodiments, the conjugate is described byformula (D-IX):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (D-IX-1):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (D-IX-2):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (D-IX-3):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (D-IX-4):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (D-IX-5):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (D-IX-6):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (D-X):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (D-X-1):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (D-X-2):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (D-X-3):

or a pharmaceutically acceptable salt thereof.

In some embodiments of any of the aspects described herein, L or L′includes one or more optionally substituted C1-C20 alkylene, optionallysubstituted C1-C20 heteroalkylene, optionally substituted C2-C20alkenylene, optionally substituted C2-C20 heteroalkenylene, optionallysubstituted C2-C20 alkynylene, optionally substituted C2-C20heteroalkynylene, optionally substituted C3-C20 cycloalkylene,optionally substituted C3-C20 heterocycloalkylene, optionallysubstituted C4-C20 cycloalkenylene, optionally substituted C4-C20heterocycloalkenylene, optionally substituted C8-C20 cycloalkynylene,optionally substituted C8-C20 heterocycloalkynylene, optionallysubstituted C5-C15 arylene, optionally substituted C2-C15 heteroarylene,O, S, NR^(i), P, carbonyl, thiocarbonyl, sulfonyl, phosphate,phosphoryl, or imino, wherein R^(i) is H, optionally substituted C1-C20alkyl, optionally substituted C1-C20 heteroalkyl, optionally substitutedC2-C20 alkenyl, optionally substituted C2-C20 heteroalkenyl, optionallysubstituted C2-C20 alkynyl, optionally substituted C2-C20 heteroalkynyl,optionally substituted C3-C20 cycloalkyl, optionally substituted C3-C20heterocycloalkyl, optionally substituted C4-C20 cycloalkenyl, optionallysubstituted C4-C20 heterocycloalkenyl, optionally substituted C8-C20cycloalkynyl, optionally substituted C8-C20 heterocycloalkynyl,optionally substituted C5-C15 aryl, or optionally substituted C2-C15heteroaryl.

In some embodiments of any of the aspects described herein, the backboneof L or L′ consists of one or more optionally substituted C1-C20alkylene, optionally substituted C1-C20 heteroalkylene, optionallysubstituted C2-C20 alkenylene, optionally substituted C2-C20heteroalkenylene, optionally substituted C2-C20 alkynylene, optionallysubstituted C2-C20 heteroalkynylene, optionally substituted C3-C20cycloalkylene, optionally substituted C3-C20 heterocycloalkylene,optionally substituted C4-C20 cycloalkenylene, optionally substitutedC4-C20 heterocycloalkenylene, optionally substituted C8-C20cycloalkynylene, optionally substituted C8-C20 heterocycloalkynylene,optionally substituted C5-C15 arylene, optionally substituted C2-C15heteroarylene, O, S, NR^(i), P, carbonyl, thiocarbonyl, sulfonyl,phosphate, phosphoryl, or imino, wherein R^(i) is H, optionallysubstituted C1-C20 alkyl, optionally substituted C1-C20 heteroalkyl,optionally substituted C2-C20 alkenyl, optionally substituted C2-C20heteroalkenyl, optionally substituted C2-C20 alkynyl, optionallysubstituted C2-C20 heteroalkynyl, optionally substituted C3-C20cycloalkyl, optionally substituted C3-C20 heterocycloalkyl, optionallysubstituted C4-C20 cycloalkenyl, optionally substituted C4-C20heterocycloalkenyl, optionally substituted C8-C20 cycloalkynyl,optionally substituted C8-C20 heterocycloalkynyl, optionally substitutedC5-C15 aryl, or optionally substituted C2-C15 heteroaryl.

In some embodiments of any of the aspects described herein, L or L′ isoxo substituted. In some embodiments, the backbone of L or L′ comprisesno more than 250 atoms. In some embodiments, L or L′ is capable offorming an amide, a carbamate, a sulfonyl, or a urea linkage. In someembodiments L or L′ is a bond. In some embodiments, L or L′ is an atom.

In some embodiments of any of the aspects described herein, each L isdescribed by formula (D-L-I):

wherein L^(A) is described by formulaG^(A1)-(Z^(A1))_(g1)-(Y^(A1))_(h1)-(Z^(A2))_(i1)-(Y^(A2))_(j1)-(Z^(A3))_(k1)-(Y^(A3))_(l1)-(Z^(A4))_(m1)-(Y^(A4))_(n1)-(Z^(A5))_(o1)-G^(A2);L^(B) is described by formulaG^(B1)-(Z^(B1))_(g2)-(Y^(B1))_(h2)-(Z^(B2))_(i2)-(Y^(B2))_(j2)-(Z^(B3))_(k2)-(Y^(B3))_(i2)-(Z^(B4))_(m2)-(Y^(B4))_(n2)-(Z^(B5))_(o2)-G^(B2);L^(C) is described by formulaG^(C1)-(Z^(C1))_(g3)-(Y^(C1))_(h3)-(Z^(C2))_(i3)-(Y^(C2))_(j3)-(Z^(C3))_(k3)-(Y^(C3))_(i3)-(Z^(C4))_(m3)-(Y^(C4))_(n3)-(Z^(C5))_(o3)-G^(C2);G^(A1) is a bond attached to Q; G^(A2) is a bond attached to A1; G^(B1)is a bond attached to Q); G^(B2) is a bond attached to A2; G^(C1) is abond attached to Q; G^(C2) is a bond attached to E or a functional groupcapable of reacting with a functional group conjugated to E (e.g.,maleimide and cysteine, amine and activated carboxylic acid, thiol andmaleimide, activated sulfonic acid and amine, isocyanate and amine,azide and alkyne, and alkene and tetrazine); each of Z^(A1), Z^(A2),Z^(A3), Z^(A4), Z^(A5), Z^(B1), Z^(B2), Z^(B3), Z^(B4), Z^(B5), Z^(C1),Z^(C2), Z^(C3), Z^(C4) and Z^(C5) is, independently, optionallysubstituted C1-C20 alkylene, optionally substituted C1-C20heteroalkylene, optionally substituted C2-C20 alkenylene, optionallysubstituted C2-C20 heteroalkenylene, optionally substituted C2-C20alkynylene, optionally substituted C2-C20 heteroalkynylene, optionallysubstituted C3-C20 cycloalkylene, optionally substituted C3-C20heterocycloalkylene, optionally substituted C4-C20 cycloalkenylene,optionally substituted C4-C20 heterocycloalkenylene, optionallysubstituted C8-C20 cycloalkynylene, optionally substituted C8-C20heterocycloalkynylene, optionally substituted C5-C15 arylene, oroptionally substituted C2-C15 heteroarylene; each of Y^(A1), Y^(A2),Y^(A3), Y^(A4), Y^(B1), Y^(B2), Y^(B3), Y^(B4), Y^(C1), Y^(C2), Y^(C3),and Y^(C4) is, independently, O, S, NR^(i), P, carbonyl, thiocarbonyl,sulfonyl, phosphate, phosphoryl, or imino; R^(i) is H, optionallysubstituted C1-C20 alkyl, optionally substituted C1-C20 heteroalkyl,optionally substituted C2-C20 alkenyl, optionally substituted C2-C20heteroalkenyl, optionally substituted C2-C20 alkynyl, optionallysubstituted C2-C20 heteroalkynyl, optionally substituted C3-C20cycloalkyl, optionally substituted C3-C20 heterocycloalkyl, optionallysubstituted C4-C20 cycloalkenyl, optionally substituted C4-C20heterocycloalkenyl, optionally substituted C8-C20 cycloalkynyl,optionally substituted C8-C20 heterocycloalkynyl, optionally substitutedC5-C15 aryl, or optionally substituted C2-C15 heteroaryl; each of g1,h1, i1, j1, k1, l1, m1, n1, o1, g2, h2, i2, j2, k2, l2, m2, n2, o2, g3,h3, i3, j3, k3, l3, m3, n3, and o3 is, independently, 0 or 1; Q is anitrogen atom, optionally substituted C1-C20 alkylene, optionallysubstituted C1-C20 heteroalkylene, optionally substituted C2-C20alkenylene, optionally substituted C2-C20 heteroalkenylene, optionallysubstituted C2-C20 alkynylene, optionally substituted C2-C20heteroalkynylene, optionally substituted C3-C20 cycloalkylene,optionally substituted C3-C20 heterocycloalkylene, optionallysubstituted C4-C20 cycloalkenylene, optionally substituted C4-C20heterocycloalkenylene, optionally substituted C8-C20 cycloalkynylene,optionally substituted C8-C20 heterocycloalkynylene, optionallysubstituted C5-C15 arylene, or optionally substituted C2-C15heteroarylene.

In some embodiments, L^(C) may have two points of attachment to the Fcdomain, Fc-binding peptide, albumin protein, or albumin protein-bindingpeptide (e.g., two G^(C2)).

In some embodiments of any of the aspects described herein, L includes apolyethylene glycol (PEG) linker. A PEG linker includes a linker havingthe repeating unit structure (—CH₂CF₂O—)_(n), wherein n is an integerfrom 2 to 100. A polyethylene glycol linker may covalently join aneuraminidase inhibitor and E (e.g., in a conjugate of any one offormulas (M-I)-(M-X)). A polyethlylene glycol linker may covalently joina first neuraminidase inhibitor and a second neuraminidase inhibitor(e.g., in a conjugate of any one of formulas (D-I)-(D-X)). Apolyethylene glycol linker may covalently joining a neuraminidaseinhibitor dimer and E (e.g., in a conjugate of any one of formulas(D-I)-(D-X)). A polyethylene glycol linker may selected any one of PEG₂to PEG₁₀₀ (e.g., PEG₂, PEG₃, PEG₄, PEG₅, PEG₅-PEG₁₀, PEG₁₀-PEG₂₀,PEG₂₀-PEG₃₀, PEG₃₀-PEG₄₀, PEG₅₀-PEG₆₀, PEG₆₀-PEG₇₀, PEG₇₀-PEG₈₀,PEG₈₀-PEG₉₀, PEG₉₀-PEG₁₀₀). In some embodiments, L^(C) includes a PEGlinker, where L^(C) is covalently attached to each of Q and E.

In some embodiments, L is

wherein z₁ and z₂ are each, independently, and integer from 1 to 20; andR₉ is selected from H, C1-C20 alkyl, C3-C20 cycloalkyl, C3-C20heterocycloalkyl; C5-C15 aryl, and C2-C15 heteroaryl.

In some embodiments, L is

wherein R* is a bond or includes one or more of optionally substitutedC1-C20 alkylene, optionally substituted C1-C20 heteroalkylene,optionally substituted C2-C20 alkenylene, optionally substituted C2-C20heteroalkenylene, optionally substituted C2-C20 alkynylene, optionallysubstituted C2-C20 heteroalkynylene, optionally substituted C3-C20cycloalkylene, optionally substituted C3-C20 heterocycloalkylene,optionally substituted C4-C20 cycloalkenylene, optionally substitutedC4-C20 heterocycloalkenylene, optionally substituted C8-C20cycloalkynylene, optionally substituted C8-C20 heterocycloalkynylene,optionally substituted C5-C15 arylene, optionally substituted C2-C15heteroarylene, O, S, NR^(i), P, carbonyl, thiocarbonyl, sulfonyl,phosphate, and imino, and wherein R^(i) is H, optionally substitutedC1-C20 alkyl, optionally substituted C1-C20 heteroalkyl, optionallysubstituted C2-C20 alkenyl, optionally substituted C2-C20 heteroalkenyl,optionally substituted C2-C20 alkynyl, optionally substituted C2-C20heteroalkynyl, optionally substituted C3-C20 cycloalkyl, optionallysubstituted C3-C20 heterocycloalkyl, optionally substituted C4-C20cycloalkenyl, optionally substituted C4-C20 heterocycloalkenyl,optionally substituted C8-C20 cycloalkynyl, optionally substitutedC8-C20 heterocycloalkynyl, optionally substituted C5-C15 aryl, oroptionally substituted C2-C15 heteroaryl.

In some embodiments, Y is:

(—NH(C═O)O—) and L is:

In some embodiments, Y is:

(—NH(C═O)O—) and L is:

In some embodiments, Y is:

(—NH(C═O)O—) and L is:

In some embodiment, Y is:

(—O—) and L is:

In another aspect, the invention features a conjugate described byformula (M-I):

wherein each A₁ is independently selected from any one of formulas(A-I)-(A-XII):

wherein R₁ is selected from —OH, —NH₂, —NHC(═NH)NH₂, and —NHC(═NH)NHR₆;R₂ and R₃ are each independently selected from —H, —OH, —F, —Cl, and—Br; R₄ is selected from —CO₂H, —P(═O)(OH)₂, —SO₃H; R₅ is selected from—COCH₃, —COCF₃, —SO₂CH₃; X is selected from —O— and —S—; Y is selectedfrom

R₆ is selected from

R₇ is selected from H, C1-C20 alkyl, C3-C20 cycloalkyl, C3-C20heterocycloalkyl; C5-C15 aryl, and C2-C15 heteroaryl; R₈ is selectedfrom C3-C20 heterocycloalkyl, C5-C15 aryl, and C2-C15 heteroaryl; each Ecomprises an Fc domain monomer (e.g., an Fc domain monomer having thesequence of any one of SEQ ID NOs: 1-68), an albumin protein (e.g., analbumin protein having the sequence of any one of SEQ ID NOs: 69-71), analbumin protein-binding peptide, or an Fc-binding peptide; n is 1 or 2;T is an integer from 1 to 20 (e.g., T is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, or 20); and L is a linker covalentlyattached to each of E and A₁, or a pharmaceutically acceptable saltthereof. When T is greater than 1 (e.g., T is 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), each A₁ may beindependently selected from any one of formulas (A-I)-(A-XII).

In another aspect, the invention features a conjugate described byformula (M-I):

wherein each A₁ is independently selected from any one of formulas(A-I)-(A-V):

wherein R₁ is selected from —OH, —NH₂, —NHC(═NH)NH₂, and —NHC(═NH)NHR₆;R₂ and R₃ are each independently selected from —H, —OH, —F, —Cl, and—Br; R₄ is selected from —CO₂H, —P(═O)(OH)₂, —SO₃H; R₅ is selected from—COCH₃, —COCF₃, —SO₂CH₃; X is selected from —O— and —S—; Y is selectedfrom

R₆ is selected from

R₇ is selected from H, C1-C20 alkyl, C3-C20 cycloalkyl, C3-C20heterocycloalkyl; C5-C15 aryl, and C2-C15 heteroaryl; R₈ is selectedfrom C3-C20 heterocycloalkyl, C5-C15 aryl, and C2-C15 heteroaryl; each Ecomprises an Fc domain monomer (e.g., an Fc domain monomer having thesequence of any one of SEQ ID NOs: 1-68), an albumin protein (e.g., analbumin protein having the sequence of any one of SEQ ID NOs: 69-71), analbumin protein-binding peptide, or an Fc-binding peptide; n is 1 or 2;T is an integer from 1 to 20 (e.g., T is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, or 20); and L is a linker covalentlyattached to each of E and A₁, or a pharmaceutically acceptable saltthereof. When T is greater than 1 (e.g., T is 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), each A₁ may beindependently selected from any one of formulas (A-I)-(A-V).

In another aspect, the invention features a conjugate described byformula (M-I):

wherein each A₁ is independently selected from any one of formulas(A-VI)-(A-IX):

wherein R₁ is selected from —OH, —NH₂, —NHC(═NH)NH₂, and —NHC(═NH)NHR₆;R₂ and R₃ are each independently selected from —H, —OH, —F, —Cl, and—Br; R₄ is selected from —CO₂H, —P(═O)(OH)₂, —SO₃H; R₅ is selected from—COCH₃, —COCF₃, —SO₂CH₃; X is selected from —O— and —S—; Y is selectedfrom

R₆ is selected from

R₇ is selected from H, C1-C20 alkyl, C3-C20 cycloalkyl, C3-C20heterocycloalkyl; C5-C15 aryl, and C2-C15 heteroaryl; R₈ is selectedfrom C3-C20 heterocycloalkyl, C5-C15 aryl, and C2-C15 heteroaryl; each Ecomprises an Fc domain monomer (e.g., an Fc domain monomer having thesequence of any one of SEQ ID NOs: 1-68), an albumin protein (e.g., analbumin protein having the sequence of any one of SEQ ID NOs: 69-71), analbumin protein-binding peptide, or an Fc-binding peptide; n is 1 or 2;T is an integer from 1 to 20 (e.g., T is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, or 20); and L is a linker covalentlyattached to each of E and A₁, or a pharmaceutically acceptable saltthereof. When T is greater than 1 (e.g., T is 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), each A₁ may beindependently selected from any one of formulas (A-VI)-(A-IX).

In some embodiments, the conjugate is described by formula (M-II):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (M-II-1):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (M-II-2):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (M-II-3):

wherein L′ is the remainder of L, and y₁ is an integer from 1-20 (e.g.,y₁ is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,or 20), or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (M-II-4):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (M-II-5):

wherein L′ is the remainder of L, and y₁ is an integer from 1-20 (e.g.,y₁ is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,or 20), or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate has the structure

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (M-II-6):

wherein R₇ is selected from H, C1-C20 alkyl, C3-C20 cycloalkyl, C3-C20heterocycloalkyl; C5-C15 aryl, and C2-C15 heteroaryl; or apharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (M-II-7):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (M-II-8):

wherein L′ is the remainder of L, and y₁ is an integer from 1-20 (e.g.,y₁ is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,or 20), or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate has the structure

In some embodiments, the conjugate is described by formula (M-II-9):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (M-II-10):

wherein L′ is the remainder of L, and y1 is an integer from 1-20 (e.g.,y1 is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,or 20), or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate has the structure

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (M-III):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (M-III-1):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (M-III-2):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (M-III-3):

wherein L′ is the remainder of L, and y₁ is an integer from 1-20 (e.g.,y₁ is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,or 20), or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (M-III-4):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (M-III-5):

wherein L′ is the remainder of L, and y₁ is an integer from 1-20 (e.g.,y₁ is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,or 20), or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (M-III-6):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (M-III-7):

wherein L′ is the remainder of L, and y₁ is an integer from 1-20 (e.g.,y₁ is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,or 20), or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (M-III-8):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (M-III-9):

wherein L′ is the remainder of L, and y₁ is an integer from 1-20 (e.g.,y₁ is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,or 20), or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (M-IV):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (M-IV-1):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (M-IV-2):

wherein L′ is the remainder of L, and y₁ is an integer from 1-20 (e.g.,y₁ is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,or 20), or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (M-V):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (M-V-1):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (M-V-2):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (M-V-3):

wherein L′ is the remainder of L, and y₁ is an integer from 1-20 (e.g.,y₁ is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,or 20), or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (M-V-4):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (M-V-5):

wherein L′ is the remainder of L, and y₁ is an integer from 1-20 (e.g.,y₁ is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,or 20), or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (M-V-6):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (M-V-7):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (M-V-8):

wherein L′ is the remainder of L, and y₁ is an integer from 1-20 (e.g.,y₁ is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,or 20), or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (M-V-9):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (M-V-10):

wherein L′ is the remainder of L, and y₁ is an integer from 1-20 (e.g.,y₁ is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,or 20), or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (M-VI):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (M-VI-1):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (M-VI-2):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (M-VI-3):

wherein L′ is the remainder of L, and y₁ is an integer from 1-20 (e.g.,y₁ is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,or 20), or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (M-VI-4):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (M-VI-5):

wherein L′ is the remainder of L, and y₁ is an integer from 1-20 (e.g.,y₁ is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,or 20), or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (M-VI-6):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (M-VI-7):

wherein L′ is the remainder of L, and y₁ is an integer from 1-20 (e.g.,y₁ is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,or 20), or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (M-VI-8):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (M-VI-9):

wherein L′ is the remainder of L, and y₁ is an integer from 1-20 (e.g.,y₁ is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,or 20), or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (M-VII):

or a pharmaceutically acceptable salt thereof.

In some embodiments of any of the aspects described herein, R₁ is OH. Insome embodiments of any of the aspects described herein, R₁ is NH₂. Insome embodiments of any of the aspects described herein, R₁ is—NHC(═NH)NH₂.

In some embodiments, the conjugate is described by formula (M-VIII):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (M-VIII-1):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by (M-VIII-2):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (M-VIII-3):

wherein L′ is the remainder of L, and y₁ is an integer from 1-20 (e.g.,y₁ is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,or 20), or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (M-VIII-4):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (M-VIII-5):

wherein L′ is the remainder of L, and y₁ is an integer from 1-20, or apharmaceutically acceptable salt thereof.

In some embodiments, the conjugate has the structure of

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (M-VIII-6):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (M-VIII-7):

wherein L′ is the remainder of L, and y₁ is an integer from 1-20 (e.g.,y₁ is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,or 20), or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (M-VIII-8):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (M-VIII-9):

wherein L′ is the remainder of L, and y₁ is an integer from 1-20 (e.g.,y₁ is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,or 20), or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (M-VIII-10):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (M-VIII-11):

wherein L′ is the remainder of L, and y₁ is an integer from 1-20 (e.g.,y₁ is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,or 20), or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (M-IX):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (M-IX-1):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (M-IX-2):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (M-IX-3):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (M-IX-4):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (M-IX-5):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (M-IX-6):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (M-X):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (M-X-1):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (M-X-2):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the conjugate is described by formula (M-X-3):

or a pharmaceutically acceptable salt thereof.

In some embodiments of any of the aspects described herein, L or L′comprises one or more optionally substituted C1-C20 alkylene, optionallysubstituted C1-C20 heteroalkylene, optionally substituted C2-C20alkenylene, optionally substituted C2-C20 heteroalkenylene, optionallysubstituted C2-C20 alkynylene, optionally substituted C2-C20heteroalkynylene, optionally substituted C3-C20 cycloalkylene,optionally substituted C3-C20 heterocycloalkylene, optionallysubstituted C4-C20 cycloalkenylene, optionally substituted C4-C20heterocycloalkenylene, optionally substituted C8-C20 cycloalkynylene,optionally substituted C8-C20 heterocycloalkynylene, optionallysubstituted C5-C15 arylene, optionally substituted C2-C15 heteroarylene,O, S, NR^(i), P, carbonyl, thiocarbonyl, sulfonyl, phosphate,phosphoryl, or imino, wherein R^(i) is H, optionally substituted C1-C20alkyl, optionally substituted C1-C20 heteroalkyl, optionally substitutedC2-C20 alkenyl, optionally substituted C2-C20 heteroalkenyl, optionallysubstituted C2-C20 alkynyl, optionally substituted C2-C20 heteroalkynyl,optionally substituted C3-C20 cycloalkyl, optionally substituted C3-C20heterocycloalkyl, optionally substituted C4-C20 cycloalkenyl, optionallysubstituted C4-C20 heterocycloalkenyl, optionally substituted C8-C20cycloalkynyl, optionally substituted C8-C20 heterocycloalkynyl,optionally substituted C5-C15 aryl, or optionally substituted C2-C15heteroaryl.

In some embodiments of any of the aspects described herein, the backboneof L or L′ consists of one or more optionally substituted C1-C20alkylene, optionally substituted C1-C20 heteroalkylene, optionallysubstituted C2-C20 alkenylene, optionally substituted C2-C20heteroalkenylene, optionally substituted C2-C20 alkynylene, optionallysubstituted C2-C20 heteroalkynylene, optionally substituted C3-C20cycloalkylene, optionally substituted C3-C20 heterocycloalkylene,optionally substituted C4-C20 cycloalkenylene, optionally substitutedC4-C20 heterocycloalkenylene, optionally substituted C8-C20cycloalkynylene, optionally substituted C8-C20 heterocycloalkynylene,optionally substituted C5-C15 arylene, optionally substituted C2-C15heteroarylene, O, S, NR^(i), P, carbonyl, thiocarbonyl, sulfonyl,phosphate, phosphoryl, or imino, wherein R^(i) is H, optionallysubstituted C1-C20 alkyl, optionally substituted C1-C20 heteroalkyl,optionally substituted C2-C20 alkenyl, optionally substituted C2-C20heteroalkenyl, optionally substituted C2-C20 alkynyl, optionallysubstituted C2-C20 heteroalkynyl, optionally substituted C3-C20cycloalkyl, optionally substituted C3-C20 heterocycloalkyl, optionallysubstituted C4-C20 cycloalkenyl, optionally substituted C4-C20heterocycloalkenyl, optionally substituted C8-C20 cycloalkynyl,optionally substituted C8-C20 heterocycloalkynyl, optionally substitutedC5-C15 aryl, or optionally substituted C2-C15 heteroaryl.

In some embodiments of any of the aspects described herein, L or L′ isoxo substituted. In some embodiments, the backbone of L or L′ comprisesno more than 250 atoms. In some embodiments, L or L′ is capable offorming an amide, a carbamate, a sulfonyl, or a urea linkage. In someembodiments, L or L′ is a bond. In some embodiments, L or L′ is an atom.In some embodiments, L′ is a nitrogen atom.

In some embodiments, each L is described by formula (M-L-1):

J¹-(Q¹)_(g)-(T¹)_(h)-(Q²)_(i)-(T²)_(j)-(Q³)_(k)-(T³)_(l)-(Q⁴)_(m)-(T⁴)_(n)-(Q⁵)_(o)-J²

wherein: J¹ is a bond attached to A1; J² is a bond attached to E or afunctional group capable of reacting with a functional group conjugatedto E (e.g., maleimide and cysteine, amine and activated carboxylic acid,thiol and maleimide, activated sulfonic acid and amine, isocyanate andamine, azide and alkyne, and alkene and tetrazine); each of Q¹, Q², Q³,Q⁴, and Q⁵ is, independently, optionally substituted C1-C20 alkylene,optionally substituted C1-C20 heteroalkylene, optionally substitutedC2-C20 alkenylene, optionally substituted C2-C20 heteroalkenylene,optionally substituted C2-C20 alkynylene, optionally substituted C2-C20heteroalkynylene, optionally substituted C3-C20 cycloalkylene,optionally substituted C3-C20 heterocycloalkylene, optionallysubstituted C4-C20 cycloalkenylene, optionally substituted C4-C20heterocycloalkenylene, optionally substituted C8-C20 cycloalkynylene,optionally substituted C8-C20 heterocycloalkynylene, optionallysubstituted C5-C15 arylene, or optionally substituted C2-C15heteroarylene; each of T¹, T², T³, T⁴ is, independently, O, S, NR^(i),P, carbonyl, thiocarbonyl, sulfonyl, phosphate, phosphoryl, or imino;R^(i) is H, optionally substituted C1-C20 alkyl, optionally substitutedC1-C20 heteroalkyl, optionally substituted C2-C20 alkenyl, optionallysubstituted C2-C20 heteroalkenyl, optionally substituted C2-C20 alkynyl,optionally substituted C2-C20 heteroalkynyl, optionally substitutedC3-C20 cycloalkyl, optionally substituted C3-C20 heterocycloalkyl,optionally substituted C4-C20 cycloalkenyl, optionally substitutedC4-C20 heterocycloalkenyl, optionally substituted C8-C20 cycloalkynyl,optionally substituted C8-C20 heterocycloalkynyl, optionally substitutedC5-C15 aryl, or optionally substituted C2-C15 heteroaryl; and each of g,h, i, j, k, l, m, n, and o is, independently, 0 or 1; or apharmaceutically acceptable salt thereof.

In some embodiments, J² may have two points of attachment to the Fcdomain, Fc-binding peptide, albumin protein, or albumin protein-bindingpeptide (e.g., two J²).

In some embodiments, L is

wherein d is an integer from 1 to 20 (e.g., d is 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20).

In some embodiments, L is

wherein each of d and e is, independently, an integer from 1 to 26; or apharmaceutically acceptable salt thereof.

In some embodiments of any of the aspects described herein, L includes apolyethylene glycol (PEG) linker. A PEG linker includes a linker havingthe repeating unit structure (—CH₂CH₂O—)_(n), wherein n is an integerfrom 2 to 100. A polyethylene glycol linker may covalently join aneuraminidase inhibitor and E (e.g., in a conjugate of any one offormulas (M-I)-(M-X)). A polyethlylene glycol linker may covalently joina first neuraminidase inhibitor and a second neuraminidase inhibitor(e.g., in a conjugate of any one of formulas (D-I)-(D-X)). Apolyethylene glycol linker may covalently join a neuraminidase inhibitordimer and E (e.g., in a conjugate of any one of formulas (D-I)-(D-X)). Apolyethylene glycol linker may selected any one of PEG₂ to PEG₁₀₀ (e.g.,PEG₂, PEG₃, PEG₄, PEG₅, PEG₅-PEG₁₀, PEG₁₀-PEG₂₀, PEG₂₀-PEG₃₀,PEG₃₀-PEG₄₀, PEG₅₀-PEG₆₀, PEG₆₀-PEG₇₀, PEG₇₀-PEG₈₀, PEG₈₀-PEG₉₀,PEG₉₀-PEG₁₀₀). In some embodiments, L^(C) includes a PEG linker, whereL^(C) is covalently attached to each of Q and E.

In some embodiments of any of the aspects described herein, R₁ is—NHC(═NH)NH₂. In some embodiments of any of the aspects describedherein, R₂ is —F. In some embodiments of any of the aspects describedherein, R₃ is —F. In some embodiments of any of the aspects describedherein, R₄ is —CO₂H. In some embodiments of any of the aspects describedherein, R₅ is —COCH₃.

In some embodiments of any of the aspects described herein, L iscovalently attached to the nitrogen atom of a surface exposed lysine ofE or L is covalently attached to the sulfur atom of a surface exposedcysteine of E.

In some embodiments of any of the aspects described herein, E is an Fcdomain monomer. In some embodiments, n is 2 and each E dimerizes to forman Fc domain.

In some embodiments, n is 2, each E is an Fc domain monomer, each Edimerizes to form an Fc domain, and the conjugate is described byformula (D-I-1):

wherein J is an Fc domain; and T is an integer from 1 to 20 (e.g., T is1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or20), or a pharmaceutically acceptable salt thereof.

In some embodiments, n is 2, each E is an Fc domain monomer, each Edimerizes to form an Fc domain, and the conjugate is described byformula (M-I-1):

wherein J is an Fc domain; and T is an integer from 1 to 20 (e.g., T is1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or20), or a pharmaceutically acceptable salt thereof.

In some embodiments of any of the aspects described herein, E has thesequence of any one of SEQ ID NOs: 1-68.

In some embodiments of any of the aspects described herein, E is analbumin protein, an albumin protein-binding peptide, or an Fc-bindingpeptide. In some embodiments, where E is an albumin protein, an albuminprotein-binding peptide, or an Fc-binding peptide, n is 1.

In some embodiments, n is 1, E is an albumin protein, an albuminprotein-binding peptide, or an Fc-binding peptide and the conjugate isdescribed by formula (D-I-2):

wherein E is an albumin protein, an albumin protein-binding peptide, orFc-binding peptide; and T is an integer from 1 to 20, or apharmaceutically acceptable salt thereof.

In some embodiments, n is 1, E is an albumin protein, an albuminprotein-binding peptide, or an Fc-binding peptide, and the conjugate isdescribed by formula (M-I-2):

wherein E is an albumin protein, an albumin protein-binding peptide, oran Fc-binding peptide; and T is an integer from 1 to 20, or apharmaceutically acceptable salt thereof.

In some embodiments of any of the aspects described herein, E is analbumin protein having the sequence of any one of SEQ ID NOs: 69-71.

In some embodiments of any of the aspects described herein, T is 1, 2,3, 4, or 5.

In another aspect, the invention provides a population of conjugateshaving the structure of any of the conjugates described herein (e.g., apopulation of conjugates having the formula of any one of formulas(1)-(5), (D-I)-(D-X), (D′-I), (M-I)-(M-X), or (M′-I)), wherein theaverage value of T is 1 to 20 (e.g., the average value of T is 1 to 2, 1to 3, 1 to 4, 1 to 5, 5 to 10, 10 to 15, or 15 to 20). In someembodiments, the average value of T is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.

In some embodiments of any of the aspects described herein, when T isgreater than 1 (e.g., T is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, or 20), each A₁-L-A₂ may be independently selected(e.g., independently selected from any of the A₁-L-A₂ structuresdescribed herein). In some embodiments, E may be conjugated to 2, 3, 4,5, 6, 7, 8, 9, 10, or more different A₁-L-A₂ moieties. In someembodiments, E is conjugated to a first A₁-L-A₂ moiety, and a secondA₁-L-A₂, moiety. In some embodiments, A₁ and A₂ of the first A₁-L-A₂moiety are independently selected from any one of formulas(A-III)-(A-V):

and A₁ and A₂ of the second A₁-L-A₂ moiety are independently selectedfrom any one of formulas (A-I), (A-II), (A-VI), (A-VII), (A-VIII), and(A-IX):

In some embodiments, each of the first A₁-L-A₂ moieties is conjugatedspecifically to a lysine residue of E (e.g., the nitrogen atom of asurface exposed lysine residue of E), and each of the second A₁-L-A₂moieties is conjugated specifically to a cysteine residue of E (e.g.,the sulfur atom of a surface exposed cysteine residue of E). In someembodiments, each of the first A₁-L-A₂ moieties is conjugatedspecifically to a cysteine residue of E (e.g., the sulfur atom of asurface exposed cysteine residue of E), and each of the second A₁-L-A₂moieties is conjugated specifically to a lysine residue of E (e.g., thenitrogen atom of a surface exposed lysine residue of E).

In some embodiments, the number of first A₁-L-A₂ moieties conjugated toE is an integer from 1 to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10).In some embodiments, the number of second A₁-L-A₂ moieties conjugated toE is an integer from 1 to 10 (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10).

In some embodiments of any of the aspects described herein, when T isgreater than 1 (e.g., T is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, or 20), each A₁-L may be independently selected(e.g., independently selected from any of the A₁-L structures describedherein). In some embodiments, E may be conjugated to 2, 3, 4, 5, 6, 7,8, 9, 10, or more different A₁-L moieties. In some embodiments, E isconjugated to a first A₁-L moiety, and a second A₁-L, moiety. In someembodiments, A₁ of the first A₁-L moiety is selected from any one offormulas (A-III)-(A-V):

and A₁ of the second A₁-L moiety is selected from any one of formulas(A-I), (A-II), (A-VI), (A-VII), (A-VIII), Of (A-IX):

In some embodiments, each of the first A₁-L moieties is conjugatedspecifically to a lysine residue of E (e.g., the nitrogen atom of asurface exposed lysine residue of E), and each of the second A₁-Lmoieties is conjugated specifically to a cysteine residue of E (e.g.,the sulfur atom of a surface exposed cysteine residue of E). In someembodiments, each of the first A₁-L moieties is conjugated specificallyto a cysteine residue of E (e.g., the sulfur atom of a surface exposedcysteine residue of E), and each of the second A₁-L moieties isconjugated specifically to a lysine residue of E (e.g., the nitrogenatom of a surface exposed lysine residue of E).

In some embodiments, the number of first A₁-L moieties conjugated to Eis an integer from 1 to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10). Insome embodiments, the number of second A₁-L moieties conjugated to E isan integer from 1 to 10 (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10).

In another aspect, the invention features a conjugate described byformula (D′-I):

wherein each A₁ is independently selected from any one of formulas(A-III)-(A-V):

wherein each A₂ is independently selected from any one of formulas(A-I), (A-II), (A-VI), (A-VII), (A-VIII), and (A-IX):

wherein R₁ is selected from —OH, —NH₂, —NHC(═NH)NH₂, and —NHC(═NH)NHR₆;R₂ and R₃ are each independently selected from —H, —OH, —F, —Cl, and—Br; R₄ is selected from —CO₂H, —P(═O)(OH)₂, —SO₃H; R₅ is selected from—COCH₃, —COCF₃, —SO₂CH₃; X is selected from —O— and —S—; Y is selectedfrom

R₆ is selected from

R₇ is selected from H, C1-C20 alkyl, C3-C20 cycloalkyl, C3-C20heterocycloalkyl; C5-C15 aryl, and C2-C15 heteroaryl; R₈ is selectedfrom C3-C20 heterocycloalkyl, C5-C15 aryl, and C2-C15 heteroaryl; each Ecomprises an Fc domain monomer (e.g., an Fc domain monomer having thesequence of any one of SEQ ID NOs: 1-68), an albumin protein (e.g., analbumin protein having the sequence of any one of SEQ ID NOs: 69-71), analbumin protein-binding peptide, or an Fc-binding peptide; n is 1 or 2;Ti is an integer from 1 to 10 (e.g., Ti is 1, 2, 3, 4, 5, 6, 7, 8, 9,10); Li is a linker covalently conjugated to E and to each A₁; T₁ is aninteger from 1 to 10 (e.g., Ti is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10); L₂ isa linker covalently conjugated to E and each A₂; T₂ is an integer from 1to 10 (e.g., T₂ is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10), or a pharmaceuticallyacceptable salt thereof.

In some embodiments, each A₁-L-A₁ is conjugated specifically to a lysineresidue of E (e.g., the nitrogen atom of a surface exposed lysineresidue of E), and each the A₂-L-A₂ is conjugated specifically to acysteine residue of E (e.g., the sulfur atom of a surface exposedcysteine residue of E). In some embodiments, each A₁-L-A₁ moiety isconjugated specifically a cysteine residue of E (e.g., the sulfur atomof a surface exposed cysteine residue of E), and each A₂-L-A₂ moiety isconjugated specifically to a lysine residue of E (e.g., the nitrogenatom of a surface exposed lysine residue of E).

In another aspect, the invention features a conjugate described byformula (M′-I):

wherein each A₁ is independently selected from any one (M-IX) offormulas (A-III)-(A-V):

wherein each A₂ is independently selected from any one of formulas(A-I), (A-II), (A-VI), (A-VII), (A-VIII), and (A-IX):

wherein R₁ is selected from —OH, —NH₂, —NHC(═NH)NH₂, and —NHC(═NH)NHR₆;R₂ and R₃ are each independently selected from —H, —OH, —F, —Cl, and—Br; R₄ is selected from —CO₂H, —P(═O)(OH)₂, —SO₃H; R₅ is selected from—COCH₃, —COCF₃, —SO₂CH₃; X is selected from —O— and —S—; Y is selectedfrom

R₆ is selected from

R₇ is selected from H, C1-C20 alkyl, C3-C20 cycloalkyl, C3-C20heterocycloalkyl; C5-C15 aryl, and C2-C15 heteroaryl; R₈ is selectedfrom C3-C20 heterocycloalkyl, C5-C15 aryl, and C2-C15 heteroaryl; each Ecomprises an Fc domain monomer (e.g., an Fc domain monomer having thesequence of any one of SEQ ID NOs: 1-68), an albumin protein (e.g., analbumin protein having the sequence of any one of SEQ ID NOs: 69-71), analbumin protein-binding peptide, or an Fc-binding peptide; n is 1 or 2;Ti is an integer from 1 to 10 (e.g., T₁ is 1, 2, 3, 4, 5, 6, 7, 8, 9,10); Li is a linker covalently conjugated to E and A₁; T₁ is an integerfrom 1 to 10 (e.g., Ti is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10); L₂ is a linkercovalently conjugated to E and A₂; T₂ is an integer from 1 to 10 (e.g.,T₂ is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10), or a pharmaceutically acceptablesalt thereof.

In some embodiments, each A₁-L is conjugated specifically to a lysineresidue of E (e.g., the nitrogen atom of a surface exposed lysineresidue of E), and each the A₂-L is conjugated specifically to acysteine residue of E (e.g., the sulfur atom of a surface exposedcysteine residue of E). In some embodiments, each A₁-L moiety isconjugated specifically a cysteine residue of E (e.g., the sulfur atomof a surface exposed cysteine residue of E), and each A₂-L moiety isconjugated specifically to a lysine residue of E (e.g., the nitrogenatom of a surface exposed lysine residue of E).

In another aspect, the invention provides a pharmaceutical compositioncomprising any of the conjugates described herein (e.g., a conjugate ofany one of formulas (1)-(5), (D-I)-(D-X), (D′-I), (M-I)-(M-X), or(M′-I)), or a pharmaceutically acceptable salt thereof, and apharmaceutically acceptable excipient.

In another aspect, the invention provides a method for the treatment ofa subject having a viral infection or presumed to have a viralinfection, the method comprising administering to the subject aneffective amount of any of the conjugates or compositions describedherein (e.g., a conjugate of any one of formulas (1)-(5), (D-I)-(D-X),(D′-I), (M-I)-(M-X), or (M′-I)).

In another aspect, the invention provides a method for the prophylactictreatment of a viral infection in a subject in need thereof, the methodcomprising administering to the subject an effective amount of any ofthe conjugates or compositions described herein (e.g., a conjugate ofany one of formulas (1)-(5), (D-I)-(D-X), (D′-I), (M-I)-(M-X), or(M′-I)).

In some embodiments, the viral infection is caused by influenza virus orparainfluenza virus. In some embodiments, the viral infection isinfluenza virus A, B, or C, or parainfluenza virus.

In some embodiments, the subject is immunocompromised.

In some embodiments, the subject has been diagnosed with humoral immunedeficiency, T cell deficiency, neutropenia, asplenia, or complementdeficiency.

In some embodiments, the subject is being treated or is about to betreated with an immunosuppresive therapy.

In some embodiments, the subject has been diagnosed with a disease whichcauses immunosuppression. In some embodiments, the disease is cancer oracquired immunodeficiency syndrome. In some embodiments, the cancer isleukemia, lymphoma, or multiple myeloma.

In some embodiments, the subject has undergone or is about to undergohematopoietic stem cell transplantation.

In some embodiments, wherein the subject has undergone or is about toundergo an organ transplant.

In some embodiments, the conjugate of composition is administeredintramuscularly, intravenously, intradermally, intraarterially,intraperitoneally, intralesionally, intracranially, intraarticularly,intraprostatically, intrapleurally, intratracheally, intranasally,intravitreally, intravaginally, intrarectally, topically,intratumorally, peritoneally, subcutaneously, subconjunctival,intravesicularlly, mucosally, intrapericardially, intraumbilically,intraocularally, orally, locally, by inhalation, by injection, or byinfusion.

In some embodiments, the subject is treated with a second therapeuticagent. In some embodiments, the second therapeutic agent is an antiviralagent. In some embodiments, the antiviral agent is selected fromoseltamivir, zanamivir, peramivir, laninamivir, amantadine, orrimantadine. In some embodiments, the second therapeutic agent is aviral vaccine. In some embodiments, the viral vaccine elicits an immuneresponse in the subject against influenza virus A, B, or C, orparainfluenza virus.

In some embodiments, an Fc-domain-containing composition may besubstituted for an Fc domain and an Fc-domain-monomer-containingcomposition may be substituted for an Fc domain monomer in any one offormulas (1)-(5), (D-I)-(D-X), (D′-I), (M-I)-(M-X), or (M′-I) (e.g., anyone of formulas (1), (2), (3), (4), (5), (D-I), (D-II), (D-II-1),(D-II-2), (D-II-3), (D-II-4), (D-II-5), (D-II-6), (D-II-7), (D-II-8),(D-II-9), (D-II-10), (D-III), (D-III-1), (D-III-2), (D-III-3),(D-III-4), (D-III-5), (D-III-6), (D-III-7), (D-III-8), (D-III-9),(D-IV), (D-IV-1), (D-IV-2), (D-V), (D-V-1), (D-V-2), (D-V-3), (D-V-4),(D-V-5), (D-V-6), (D-V-7), (D-V-8), (D-V-9), (D-V-10), (D-VI), (D-VI-1),(D-VI-2), (D-VI-3), (D-VI-4), (D-VI-5), (D-VI-6), (D-VI-7), (D-VI-8),(D-VI-9), (D-VII), (D-VIII), (D-VIII-1), (D-VIII-2), (D-VIII-3),(D-VIII-4), (D-VIII-5), (D-VIII-6), (D-VIII-7), (D-VIII-8), (D-VIII-9),(D-VIII-10), (D-VIII-11), (D-IX), (D-IX-1), (D-IX-2), (D-IX-3),(D-IX-4), (D-IX-5), (D-IX-8), (D-X), (D-X-1), (D-X-2), (D-X-3), (D′-I),(M-I), (M-II), (M-II-1), (M-II-2), (M-II-3), (M-II-4), (M-II-5),(M-II-6), (M-II-7), (M-II-8), (M-II-9), (M-II-10), (M-III), (M-III-1),(M-III-2), (M-III-3), (M-III-4), (M-III-5), (M-III-6), (M-III-7),(M-III-8), (M-III-9), (M-IV), (M-IV-1), (M-IV-2), (M-V), (M-V-1),(M-V-2), (M-V-3), (M-V-4), (M-V-5), (M-V-6), (M-V-7), (M-V-8), (M-V-9),(M-V-10), (M-VI), (M-VI-1), (M-VI-2), (M-VI-3), (M-VI-4), (M-VI-5),(M-VI-6), (M-VI-7), (M-VI-8), (M-VI-9), (M-VII), (M-VIII), (M-VIII-1),(M-VIII-2), (M-VIII-3). (M-VIII-4), (M-VIII-5), (M-VIII-6), (M-VIII-7),(M-VIII-8). (M-VIII-9), (M-VIII-10), (M-VIII-11), (M-IX), (M-IX-1),(M-IX-2), (M-IX-3), (M-IX-4), (M-IX-5), (M-IX-6), (M-X), (M-X-1),(M-X-2), (M-X-3), or (M′-I)). In any of the formulas described herein(e.g., any one of formulas (1)-(5), (D-I)-(D-X), (D′-I), (M-I)-(M-X), or(M′-I)), when n is 1, E is an Fc-domain-monomer-containing composition,in any of the formulas described herein (e.g., any one of formulas(1)-(5), (D-I)-(D-X), (D′-I), (M-I)-(M-X), or (M′-I)), when n is 2, E isan Fc-domain-containing composition.

In certain embodiments, the Fc-domain-containing composition is anantibody or an antibody fragment. An antibody may include any form ofimmunoglobulin, heavy chain antibody, light chain antibody, LRR-basedantibody, or other protein scaffold with antibody-like properties, aswell as any other immunological binding moiety known in the art,including antibody fragments (e.g., a Fab, Fab′, Fab′2, F(ab′)2, Fd,F(ab′)2, Fd, Fv, Feb, scFv, or SMIP). The subunit structures andthree-dimensional configurations of different classes of antibodies areknown in the art. An antibody fragment may include a binding moiety thatincludes a portion derived from or having significant homology to anantibody, such as the antigen-determining region of an antibody.Exemplary antibody fragments include Fab, Fab′, Fab′2, F(ab′)2, Fd, Fv,Feb, scFv, and SMIP.

In particular embodiments, the antibody or antibody fragment is a human,mouse, camelid (e.g., llama, alpaca, or camel), goat, sheep, rabbit,chicken, guinea pig, hamster, horse, or rat antibody or antibodyfragment. In specific embodiments, the antibody is an IgG, IgA, IgD,IgE, IgM, or intrabody. In certain embodiments, the antibody fragmentincludes an scFv, sdAb, dAb, Fab, Fab′, Fab′2, F(ab′)2, Fd, Fv, Feb, orSMIP.

In some embodiments, the Fc-domain-containing composition (e.g., anantibody or antibody fragment) confers binding specificity to a one ormore targets (e.g., an antigen). in some embodiments, the one or moretargets (e.g., an antigen) bound by the Fc-domain-containing composition(e.g., an antibody or antibody fragment) is a viral (e.g., influenza)protein such as neuraminidase or hemagglutinin. In some embodiments, theantibody or antibody fragment recognizes a viral surface antigen. Insome embodiments, the antibody or antibody fragment targetshemagglutinin. Hemagglutinin-targeting antibodies include monoclonalantibodies, such as CR8281, CR8020, MEDI8852, MHAA4549A, and VIS410. Insome embodiments, the antibody or antibody fragment is a broadlyneutralizing antibody or antibody fragment targeting influenzahemagglutinin (e.g., an antibody or antibody fragment described in Wu etal., J. Mol. Biol. 429:2694-2709 (2017)). in some embodiments theantibody or antibody fragment targets a viral matrix protein (e.g.,matrix 2 protein). TCN032 is a matrix 2 protein targeting monoclonalantibody.

In some embodiments, the Fc-domain-containing composition (e.g., anantibody or antibody fragment) includes one or more single-domainantibodies (sdAbs). In some embodiments the Fc-domain-containingcomposition is an antibody or antibody fragment including a sdAb withinfluenza A reactivity, such as a sdAb that binds to hemagglutinin ofinfluenza A (e.g., SD36 or SD38, described in Laursen et ai. Science.362:598-602 (2018)). In some embodiments the Fc-domain-containingcomposition is an antibody or antibody fragment including a sdAb withinfluenza B reactivity, such as a sdAb that binds to hemagglutinin ofinfluenza B (e.g., SD83 or SD84, described in Laursen et ai. Science.362:598-602 (2018)).

In some embodiments the Fc-domain-containing composition is amultidomain antibody (MDAb) or multidomain antibody fragment including 2or more (e.g., 2, 3, 4, 5, 8, 7, 8, 9 or 10 or more) sdAbs. in someembodiments, the MDAb or fragment thereof includes one or more sdABsthat binds to hemaglutinin of influenza A, and one or more sdAbs thatbinds to hemagglutinin of influenza B. In some embodiments, the MDAb isJNJ-7445 (also known as MD38Q8), which is described in Laursen et al.Science. 382:598-802 (2018). In brief, JNJ-7445 is an MDAb that includestwo sdAbs that bind to hemagglutinin of influenza A (SD38 and SD38) andtwo sdAbs that bind to hemagglutinin of influenza B (SD83 and SD84),which are linked to an Fc domain (IgG1). The sdAbs were produced byimmunizing llamas with influenza vaccine and H7 and H2 recombinanthemagglutinin.

In another aspect, the invention includes a conjugate described by anyone of formulas (1)-(5), (D-I)-(D-X), (D′-I), (M-I)-(M-X), or (M′-I)(e.g., anyone of formulas (1), (2), (3), (4), (5), (D-I), (D-II),(D-II-1), (D-II-2), (D-II-3), (D-II-4), (D-II-5), (D-II-6), (D-II-7),(D-II-8), (D-II-9), (D-II-10), (D-III), (D-III-1), (D-III-2), (D-III-3),(D-III-4), (D-III-5), (D-III-6), (D-III-7), (D-III-8), (D-III-9),(D-IV), (D-IV-1), (D-IV-2), (D-V), (D-V-1), (D-V-2), (D-V-3), (D-V-4),(D-V-5), (D-V-6), (D-V-7), (D-V-8), (D-V-9), (D-V-10), (D-VI), (D-VI-1),(D-VI-2), (D-VI-3), (D-VI-4), (D-VI-5), (D-VI-6), (D-VI-7), (D-VI-8),(D-VI-9), (D-VII), (D-VIII), (D-VIII-1), (D-VIII-2), (D-VIII-3),(D-VIII-4), (D-VIII-5), (D-VIII-6), (D-VIII-7), (D-VIII-8), (D-VIII-9),(D-VIII-10), (D-VIII-11), (D-IX), (D-IX-1), (D-IX-2), (D-IX-3),(D-IX-4), (D-IX-5), (D-IX-6), (D-X), (D-X-1), (D-X-2), (D-X-3), (D′-I),(M-I), (M-II), (M-II-1), (M-II-2), (M-II-3), (M-II-4), (M-II-5),(M-II-6), (M-II-7), (M-II-8), (M-II-9), (M-II-10), (M-III), (M-III-1),(M-III-2), (M-III-3), (M-III-4), (M-III-5), (M-III-6), (M-III-7),(M-III-8), (M-III-9), (M-IV), (M-IV-1), (M-IV-2), (M-V), (M-V-1),(M-V-2), (M-V-3), (M-V-4), (M-V-5), (M-V-6), (M-V-7), (M-V-8), (M-V-9),(M-V-10), (M-VI), (M-VI-1), (M-VI-2), (M-VI-3), (M-VI-4), (M-VI-5),(M-VI-6), (M-VI-7), (M-VI-8), (M-VI-9), (M-VII), (M-VIII), (M-VIII-1),(M-VIII-2), (M-VIII-3), (M-VIII-4), (M-VIII-5), (M-VIII-6), (M-VIII-7),(M-VIII-8), (M-VIII-9), (M-VIII-10), (M-VIII-11), (M-IX), (M-IX-1),(M-IX-2), (M-IX-3), (M-IX-4), (M-IX-5), (M-IX-6), (M-X), (M-X-1),(M-X-2), (M-X-3), or (M′-I)), where E is an antibody or antibodyfragment, in preferred embodiments, where E is an antibody or antibodyfragment, n is 1. In some embodiments, the antibody or antibody fragmentincludes any antibody or antibody fragment described herein, such as amonoclonal antibody that binds to viral hemagglutinin (e.g., CR6261,CR8020, MEDI8852, MHAA4549A, or VIS410); a broadly neutralizing antibodyor antibody fragment targeting viral hemagglutinin (e.g., antibodies orantibody fragments described in Wu et al., J. Mol. Biol. 429:2894-2709(2017)); a sdAb targeting viral hemagglutinin (e.g., SD36, SD38, SD83,or SD84); or a MDAb or fragment thereof targeting viral hemaglutinin(e.g., JNJ-7445).

In some embodiments of any of the aspects described herein, E (e.g.,each E) includes the amino acid sequence of SEQ ID NO: 1. in someembodiments, E includes an amino acid sequence that is at least 70%,75%, 80%, 85%, 90%, 95%, 98%, 97%, 98%, 99%, or 100% identical to theamino acid sequence of SEQ ID NO: 1.

In some embodiments of any of the aspects described herein, E (e.g.,each E) includes the amino acid sequence of SEQ ID NO: 2, in someembodiments, E includes an amino acid sequence that is at least 70%,75%, 80%, 85%, 90%, 95%, 98%, 97%, 98%, 99%, or 100% identical to theamino acid sequence of SEQ ID NO: 2.

In some embodiments of any of the aspects described herein, E (e.g.,each E) includes the amino acid sequence of SEQ ID NO: 3. in someembodiments, E includes an amino acid sequence that is at least 70%,75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to theamino acid sequence of SEQ ID NO: 3.

In some embodiments of any of the aspects described herein, E (e.g.,each E) includes the amino acid sequence of SEQ ID NO: 4. In someembodiments, E includes an amino acid sequence that is at least 70%,75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to theamino acid sequence of SEQ ID NO: 4.

In some embodiments of any of the aspects described herein, E (e.g.,each E) includes the amino acid sequence of SEQ ID NO: 5. In someembodiments, E includes an amino acid sequence that is at least 70%,75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to theamino acid sequence of SEQ ID NO: 5.

In some embodiments of any of the aspects described herein, E (e.g.,each E) includes the amino acid sequence of SEQ ID NO: 6. in someembodiments, E includes an amino acid sequence that, is at least 70%,75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to theamino acid sequence of SEQ ID NO: 6.

In some embodiments of any of the aspects described herein, E (e.g.,each E) includes the amino acid sequence of SEQ ID NO: 7. in someembodiments, E includes an amino acid sequence that, is at least 70%,75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to theamino acid sequence of SEQ ID NO: 7.

In some embodiments of any of the aspects described herein, E (e.g.,each E) includes the amino acid sequence of SEQ ID NO: 8. in someembodiments, E includes an amino acid sequence that is at least 70%,75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to theamino acid sequence of SEQ ID NO: 8.

In some embodiments of any of the aspects described herein, E (e.g.,each E) includes the amino acid sequence of SEQ ID NO: 9. in someembodiments, E includes an amino acid sequence that is at least 70%,75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to theamino acid sequence of SEQ ID NO: 9.

In some embodiments of any of the aspects described herein, E (e.g.,each E) includes the amino acid sequence of SEQ ID NO: 10. in someembodiments, E includes an amino acid sequence that is at least 70%,75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to theamino acid sequence of SEQ ID NO: 10.

In some embodiments of any of the aspects described herein, E (e.g.,each E) includes the amino acid sequence of SEQ ID NO: 11. In someembodiments, E includes an amino acid sequence that is at least 70%,75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to theamino acid sequence of SEQ ID NO: 11.

In some embodiments of any of the aspects described herein, E (e.g.,each E) includes the amino acid sequence of SEQ ID NO: 12. In someembodiments, E includes an amino acid sequence that is at least 70%,75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to theamino acid sequence of SEQ ID NO: 12.

In some embodiments of any of the aspects described herein, E (e.g.,each E) includes the amino acid sequence of SEQ ID NO: 13. In someembodiments, E includes an amino acid sequence that is at least 70%,75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to theamino acid sequence of SEQ ID NO: 13.

In some embodiments of any of the aspects described herein, E (e.g.,each E) includes the amino acid sequence of SEQ ID NO: 14. In someembodiments, E includes an amino acid sequence that is at least 70%,75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to theamino acid sequence of SEQ ID NO: 14.

In some embodiments of any of the aspects described herein, E (e.g.,each E) includes the amino acid sequence of SEQ ID NO: 15. In someembodiments, E includes an amino acid sequence that is at least 70%,75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to theamino acid sequence of SEQ ID NO: 15.

In some embodiments of any of the aspects described herein, E (e.g.,each E) includes the amino acid sequence of SEQ ID NO: 16. In someembodiments, E includes an amino acid sequence that, is at least 70%,75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to theamino acid sequence of SEQ ID NO: 16.

In some embodiments of any of the aspects described herein, E (e.g.,each E) includes the amino acid sequence of SEQ ID NO: 17. In someembodiments, E includes an amino acid sequence that is at least 70%,75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to theamino acid sequence of SEQ ID NO: 17.

In some embodiments of any of the aspects described herein, E (e.g.,each E) includes the amino acid sequence of SEQ ID NO: 18. in someembodiments, E includes an amino acid sequence that is at least 70%,75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to theamino acid sequence of SEQ ID NO: 18.

In some embodiments of any of the aspects described herein, E (e.g.,each E) includes the amino acid sequence of SEQ ID NO: 19. in someembodiments, E includes an amino acid sequence that is at least 70%,75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to theamino acid sequence of SEQ ID NO: 19.

In some embodiments of any of the aspects described herein, E (e.g.,each E) includes the amino acid sequence of SEQ ID NO: 20. In someembodiments, E includes an amino acid sequence that is at least 70%,75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to theamino acid sequence of SEQ ID NO: 20.

In some embodiments of any of the aspects described herein, E (e.g.,each E) includes the amino acid sequence of SEQ ID NO: 21. In someembodiments, E includes an amino acid sequence that is at least 70%,75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to theamino acid sequence of SEQ ID NO: 21.

In some embodiments of any of the aspects described herein, E (e.g.,each E) includes the amino acid sequence of SEQ ID NO: 22. In someembodiments, E includes an amino acid sequence that is at least 70%,75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%. identical to theamino acid sequence of SEQ ID NO: 22.

In some embodiments of any of the aspects described herein, E (e.g.,each E) includes the amino acid sequence of SEQ ID NO: 23. In someembodiments, E includes an amino acid sequence that is at least 70%,75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to theamino acid sequence of SEQ ID NO: 23.

In some embodiments of any of the aspects described herein, E (e.g.,each E) includes the amino acid sequence of SEQ ID NO: 24. In someembodiments, E includes an amino acid sequence that is at least 70%,75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to theamino acid sequence of SEQ ID NO: 24.

In some embodiments of any of the aspects described herein, E (e.g.,each E) includes the amino acid sequence of SEQ ID NO: 25. In someembodiments, E includes an amino acid sequence that is at least 70%,75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to theamino acid sequence of SEQ ID NO: 25.

In some embodiments of any of the aspects described herein, E (e.g.,each E) includes the amino acid sequence of SEQ ID NO: 26. In someembodiments, E includes an amino acid sequence that, is at least 70%,75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to theamino acid sequence of SEQ ID NO: 26.

In some embodiments of any of the aspects described herein, E (e.g.,each E) includes the amino acid sequence of SEQ ID NO: 27. In someembodiments, E includes an amino acid sequence that is at least 70%,75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to theamino acid sequence of SEQ ID NO: 27.

In some embodiments of any of the aspects described herein, E (e.g.,each E) includes the amino acid sequence of SEQ ID NO: 28. in someembodiments, E includes an amino acid sequence that is at least 70%,75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to theamino acid sequence of SEQ ID NO: 28.

In some embodiments of any of the aspects described herein, E (e.g.,each E) includes the amino acid sequence of SEQ ID NO: 29. in someembodiments, E includes an amino acid sequence that is at least 70%,75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to theamino acid sequence of SEQ ID NO: 29.

In some embodiments of any of the aspects described herein, E (e.g.,each E) includes the amino acid sequence of SEQ ID NO: 30. In someembodiments, E includes an amino acid sequence that is at least 70%,75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to theamino acid sequence of SEQ ID NO: 30.

In some embodiments of any of the aspects described herein, E (e.g.,each E) includes the amino acid sequence of SEQ ID NO: 31. In someembodiments, E includes an amino acid sequence that is at least 70%,75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to theamino acid sequence of SEQ ID NO: 31.

In some embodiments of any of the aspects described herein, E (e.g.,each E) includes the amino acid sequence of SEQ ID NO: 32. In someembodiments, E includes an amino acid sequence that is at least 70%,75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to theamino acid sequence of SEQ ID NO: 32.

In some embodiments of any of the aspects described herein, E (e.g.,each E) includes the amino acid sequence of SEQ ID NO: 33. In someembodiments, E includes an amino acid sequence that is at least 70%,75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to theamino acid sequence of SEQ ID NO: 33.

In some embodiments of any of the aspects described herein, E (e.g.,each E) includes the amino acid sequence of SEQ ID NO: 34. In someembodiments, E includes an amino acid sequence that is at least 70%,75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to theamino acid sequence of SEQ ID NO: 34.

In some embodiments of any of the aspects described herein, E (e.g.,each E) includes the amino acid sequence of SEQ ID NO: 35. In someembodiments, E includes an amino acid sequence that is at least 70%,75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to theamino acid sequence of SEQ ID NO: 35.

In Some Embodiments of any of the Aspects Described Herein, E (e.g.,Each E) Includes the Amino acid sequence of SEQ ID NO: 36. In someembodiments, E includes an amino acid sequence that, is at least 70%,75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to theamino acid sequence of SEQ ID NO: 36.

In some embodiments of any of the aspects described herein, E (e.g.,each E) includes the amino acid sequence of SEQ ID NO: 37. In someembodiments, E includes an amino acid sequence that is at least 70%,75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to theamino acid sequence of SEQ ID NO: 37.

In some embodiments of any of the aspects described herein, E (e.g.,each E) includes the amino acid sequence of SEQ ID NO: 38. in someembodiments, E includes an amino acid sequence that is at least 70%,75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to theamino acid sequence of SEQ ID NO: 38.

In some embodiments of any of the aspects described herein, E (e.g.,each E) includes the amino acid sequence of SEQ ID NO: 39. in someembodiments, E includes an amino acid sequence that is at least 70%,75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to theamino acid sequence of SEQ ID NO: 39.

In some embodiments of any of the aspects described herein, E (e.g.,each E) includes the amino acid sequence of SEQ ID NO: 40. In someembodiments, E includes an amino acid sequence that is at least 70%,75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to theamino acid sequence of SEQ ID NO: 40.

In some embodiments of any of the aspects described herein, E (e.g.,each E) includes the amino acid sequence of SEQ ID NO: 41. In someembodiments, E includes an amino acid sequence that is at least 70%,75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to theamino acid sequence of SEQ ID NO: 41.

In some embodiments of any of the aspects described herein, E (e.g.,each E) includes the amino acid sequence of SEQ ID NO: 42. In someembodiments, E includes an amino acid sequence that is at least 70%,75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%. identical to theamino acid sequence of SEQ ID NO: 42.

In some embodiments of any of the aspects described herein, E (e.g.,each E) includes the amino acid sequence of SEQ ID NO: 43. In someembodiments, E includes an amino acid sequence that is at least 70%,75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to theamino acid sequence of SEQ ID NO: 43.

In some embodiments of any of the aspects described herein, E (e.g.,each E) includes the amino acid sequence of SEQ ID NO: 44. In someembodiments, E includes an amino acid sequence that is at least 70%,75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to theamino acid sequence of SEQ ID NO: 45.

In some embodiments of any of the aspects described herein, E (e.g.,each E) includes the amino acid sequence of SEQ ID NO: 46. In someembodiments, E includes an amino acid sequence that is at least 70%,75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to theamino acid sequence of SEQ ID NO: 46.

In some embodiments of any of the aspects described herein, E (e.g.,each E) includes the amino acid sequence of SEQ ID NO: 47. In someembodiments, E includes an amino acid sequence that, is at least 70%,75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to theamino acid sequence of SEQ ID NO: 47.

In some embodiments of any of the aspects described herein, E (e.g.,each E) includes the amino acid sequence of SEQ ID NO: 48. In someembodiments, E includes an amino acid sequence that is at least 70%,75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to theamino acid sequence of SEQ ID NO: 48.

In some embodiments of any of the aspects described herein, E (e.g.,each E) includes the amino acid sequence of SEQ ID NO: 49. in someembodiments, E includes an amino acid sequence that is at least 70%,75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to theamino acid sequence of SEQ ID NO: 49.

In some embodiments of any of the aspects described herein, E (e.g.,each E) includes the amino acid sequence of SEQ ID NO: 50. in someembodiments, E includes an amino acid sequence that is at least 70%,75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to theamino acid sequence of SEQ ID NO: 50.

In some embodiments of any of the aspects described herein, E (e.g.,each E) includes the amino acid sequence of SEQ ID NO: 51. In someembodiments, E includes an amino acid sequence that is at least 70%,75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to theamino acid sequence of SEQ ID NO: 51.

In some embodiments of any of the aspects described herein, E (e.g.,each E) includes the amino acid sequence of SEQ ID NO: 52. In someembodiments, E includes an amino acid sequence that is at least 70%,75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to theamino acid sequence of SEQ ID NO: 52.

In some embodiments of any of the aspects described herein, E (e.g.,each E) includes the amino acid sequence of SEQ ID NO: 53. In someembodiments, E includes an amino acid sequence that is at least 70%,75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%. identical to theamino acid sequence of SEQ ID NO: 53.

In some embodiments of any of the aspects described herein, E (e.g.,each E) includes the amino acid sequence of SEQ ID NO: 54. In someembodiments, E includes an amino acid sequence that is at least 70%,75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to theamino acid sequence of SEQ ID NO: 54.

In some embodiments of any of the aspects described herein, E (e.g.,each E) includes the amino acid sequence of SEQ ID NO: 55. In someembodiments, E includes an amino acid sequence that is at least 70%,75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to theamino acid sequence of SEQ ID NO: 55.

In some embodiments of any of the aspects described herein, E (e.g.,each E) includes the amino acid sequence of SEQ ID NO: 56. In someembodiments, E includes an amino acid sequence that is at least 70%,75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to theamino acid sequence of SEQ ID NO: 56.

In some embodiments of any of the aspects described herein, E (e.g.,each E) includes the amino acid sequence of SEQ ID NO: 57. In someembodiments, E includes an amino acid sequence that, is at least 70%,75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to theamino acid sequence of SEQ ID NO: 57.

In some embodiments of any of the aspects described herein, E (e.g.,each E) includes the amino acid sequence of SEQ ID NO: 58. In someembodiments, E includes an amino acid sequence that is at least 70%,75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to theamino acid sequence of SEQ ID NO: 58.

In some embodiments of any of the aspects described herein, E (e.g.,each E) includes the amino acid sequence of SEQ ID NO: 59. in someembodiments, E includes an amino acid sequence that is at least 70%,75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to theamino acid sequence of SEQ ID NO: 59.

In some embodiments of any of the aspects described herein, E (e.g.,each E) includes the amino acid sequence of SEQ ID NO: 60. in someembodiments, E includes an amino acid sequence that is at least 70%,75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to theamino acid sequence of SEQ ID NO: 60.

In some embodiments of any of the aspects described herein, E (e.g.,each E) includes the amino acid sequence of SEQ ID NO: 61. In someembodiments, E includes an amino acid sequence that is at least 70%,75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to theamino acid sequence of SEQ ID NO: 61.

In some embodiments of any of the aspects described herein, E (e.g.,each E) includes the amino acid sequence of SEQ ID NO: 62. In someembodiments, E includes an amino acid sequence that is at least 70%,75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to theamino acid sequence of SEQ ID NO: 62.

In some embodiments of any of the aspects described herein, E (e.g.,each E) includes the amino acid sequence of SEQ ID NO: 63. In someembodiments, E includes an amino acid sequence that is at least 70%,75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%. identical to theamino acid sequence of SEQ ID NO: 63.

In some embodiments of any of the aspects described herein, E (e.g.,each E) includes the amino acid sequence of SEQ ID NO: 84. In someembodiments, E includes an amino acid sequence that is at least 70%,75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to theamino acid sequence of SEQ ID NO: 84.

In some embodiments of any of the aspects described herein, E (e.g.,each E) includes the amino acid sequence of SEQ ID NO: 85. In someembodiments, E includes an amino acid sequence that is at least 70%,75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to theamino acid sequence of SEQ ID NO: 85.

In some embodiments of any of the aspects described herein, E (e.g.,each E) includes the amino acid sequence of SEQ ID NO: 86. In someembodiments, E includes an amino acid sequence that is at least 70%,75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to theamino acid sequence of SEQ ID NO: 86.

In some embodiments of any of the aspects described herein, E (e.g.,each E) includes the amino acid sequence of SEQ ID NO: 67. In someembodiments, E includes an amino acid sequence that, is at least 70%,75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to theamino acid sequence of SEQ ID NO: 67.

In some embodiments of any of the aspects described herein, E (e.g.,each E) includes the amino acid sequence of SEQ ID NO: 68. In someembodiments, E includes an amino acid sequence that is at least 70%,75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to theamino acid sequence of SEQ ID NO: 68.

In some embodiments of any of the aspects described herein, E (e.g.,each E) includes the amino acid sequence of SEQ ID NO: 69. in someembodiments, E includes an amino acid sequence that is at least 70%,75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to theamino acid sequence of SEQ ID NO: 69.

In some embodiments of any of the aspects described herein, E (e.g.,each E) includes the amino acid sequence of SEQ ID NO: 70. in someembodiments, E includes an amino acid sequence that is at least 70%,75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to theamino acid sequence of SEQ ID NO: 70.

In some embodiments of any of the aspects described herein, E (e.g.,each E) includes the amino acid sequence of SEQ ID NO: 71. In someembodiments, E includes an amino acid sequence that is at least 70%,75%, 80%, 85%, 90%, 95%, 98%, 97%, 98%, 99%, or 100% identical to theamino acid sequence of SEQ ID NO: 71.

In some embodiments of any of the aspects described herein, wherein Eincludes an Fc domain monomer, the Fc domain monomer (e.g., the Fcdomain monomer having the sequence of any one of SEQ ID NOs: 1-68)includes a triple mutation corresponding to M252Y/S254T/T256E (YTE). Asused herein, an amino acid “corresponding to” a particular amino acidresidue (e.g., of a particular SEQ ID NO.) should be understood toinclude any amino acid residue that one of skill in the art wouldunderstand to align to the particular residue (e.g., of the particularsequence). For example, any one of SEQ ID NOs: 1-68 may be mutated toinclude a YTE mutation.

In some embodiments of any of the aspects described herein, wherein Eincludes an Fc domain monomer, the Fc domain monomer (e.g., the Fcdomain monomer having the sequence of any one of SEQ ID NOs: 1-68)includes a double mutant corresponding to M428L/N434S (LS). As usedherein, an amino acid “corresponding to” a particular amino acid residue(e.g., of a particular SEQ ID NO.) should be understood to include anyamino acid residue that one of skill in the art would understand toalign to the particular residue (e.g., of the particular sequence). Forexample, anyone of SEQ ID NOs: 1-68 may be mutated to include a LSmutation.

In some embodiments of any of the aspects described herein, wherein Eincludes an Fc domain monomer, the Fc domain monomer (e.g., the Fcdomain monomer having the sequence of any one of SEQ ID NOs: 1-68)includes a mutant corresponding to N434H. As used herein, an amino acid“corresponding to” a particular amino acid residue (e.g., of aparticular SEQ ID NO.) should be understood to include any amino acidresidue that one of skill in the art would understand to align to theparticular residue (e.g., of the particular sequence). For example,anyone of SEQ ID NOs: 1-68 may be mutated to include an N434H mutation.

In some embodiments of any of the aspects described herein, wherein Eincludes an Fc domain monomer, the Fc domain monomer (e.g., the Fcdomain monomer having the sequence of any one of SEQ ID NOs: 1-68)includes a mutant corresponding to C220S. As used herein, an amino acid“corresponding to” a particular amino acid residue (e.g., of aparticular SEQ ID NO.) should be understood to include any amino acidresidue that one of skill in the art would understand to align to theparticular residue (e.g., of the particular sequence). For example,anyone of SEQ ID NOs: 1-68 may be mutated to include a C220S mutation.

In some embodiments of any of the aspects described herein, wherein Eincludes an Fc domain monomer, the Fc domain monomer (e.g., the Fcdomain monomer having the sequence of any one of SEQ ID NOs: 1-68) is afragment of the Fc domain monomer (e.g., a fragment of at least 25(e.g., 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more), atleast 50 (e.g., 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64,65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75 or more), at least 75 (e.g.,75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92,93, 94, 95, 96, 97, 98, 99, 100 or more) consecutive amino acids inlength from SEQ ID NOs: 1-68.

In some embodiments of any of the aspects described herein (e.g., aconjugate of any one of formulas (1)-(5), (D-I)-(D-X), (D′-I),(M-I)-(M-X), or (M′-I)), one or more nitrogen atoms of one or moresurface exposed lysine residues of E or one or more sulfur atoms of oneor more surface exposed cysteines in E is covalently conjugated to alinker (e.g., a PEG₂-PEG₂₀ linker). The linker conjugated to E may befunctionalized such that it may reacts to form a covalent bond with theL of any A₁-L or any A₂-L-A₁ described herein. In preferred embodiments,E is conjugated to a linker functionalized with an azido group and the Lof A₁-L or any A₂-L-A₁ is functionalized with an alkyne group.Conjugation (e.g., by click chemistry) of the linker-azido of E andlinker-alkyne of A₁-L or A₂-L-A₁ forms a conjugate of the invention, forexample a conjugate described by any one of formulas (1)-(5),(D-I)-(D-X), (D′-I), (M-I)-(M-X), or (M′-I). In yet other embodiments, Eis conjugated to a linker functionalized with an alkyne group and L ofany A₁-L or of any A₂-L-A₁ is functionalized with an azido group.Conjugation (e.g., by click chemistry) of the linker-alkyne of E andlinker-azido of A₁-L or A₂-L-A₁ forms a conjugate of the invention, forexample a conjugate described by formula (1)-(5), (D-I)-(D-X), (D′-I),(M-I)-(M-X), or (M′-I).

In some embodiments of any of the aspects described herein, the squigglyline of any one of formulas (1)-(5), (D-I)-(D-X), (D′-I), (M-I)-(M-X),or (M′-I) may represent a covalent bond between E and the L of A₁-L orA₂-L-A₁.

In some embodiments of any of the aspects described herein, the squigglyline of any one of formulas (1)-(5), (D-I)-(D-X), (D′-I), (M-I)-(M-X),or (M′-I) may represent that one or more amino acid side chains of E(e.g., one or more nitrogen atoms of one or more surface exposed lysineresidues of E or one or more sulfur atoms of one or more surface exposedcysteines in E) have been conjugated to a linker (e.g., a PEG₂-PEG₂₀linker) wherein the linker has been functionalized with a reactivemoiety, such that the reactive moiety forms a covalent bond with the Lof any A₁-L or any A₂-L-A₁ described herein (e.g., by click chemistrybetween an azido functionalized linker and an alkyne functionalizedlinker, as described above). In some embodiments of any of the aspectsdescribed herein, A₁ and/or A₂ have the structure described by (A-I):

In preferred embodiments, wherein A₁ and/or A₂ have the structuredescribed by (A-I): R₁ is —NHC(═NH)NH₂, R₄ is —CO₂H, R₅ is —COCH₃,and/or X is —O—. In preferred embodiments, A₁ and/or A₂ have thestructure of zanamivir described by:

In some embodiments of any of the aspects described herein, A₁ and/or A₂have the structure described by (A-II):

In preferred embodiments, wherein A₁ and/or A₂ have the structuredescribed by (A-II): R₁ is —NHC(═NH)NH₂, R₂ is H or F, R₃ is H or F, R₄is —CO₂H, R₅ is —COCH₃, and/or X is —O—. In preferred embodiments, A₁and/or A₂ have the structure described by:

In some embodiments of any of the aspects described herein, A₁ and/or A₂have the structure described by (A-III):

In preferred embodiments, wherein A₁ and/or A₂ have the structuredescribed by (A-III): R₁ is —NHC(═NH)NH₂, R₄ is —CO₂H, and/or R₅ is—COCH₃. In preferred embodiments, A₁ and/or A₂ have the structure ofperamivir described by:

In some embodiments of any of the aspects described herein, A₁ and/or A₂have the structure described by (A-IV):

In preferred embodiments, wherein A₁ and/or A₂ have the structuredescribed by (A-IV): R₁ is —NHC(═NH)NH₂, R₄ is —CO₂H, and/or R₅ is—COCH₃. In preferred embodiments, A₁ and/or A₂ have the structuredescribed by:

In some embodiments of any of the aspects described herein, A₁ and/or A₂have the structure described by (A-V):

In preferred embodiments, wherein A₁ and/or A₂ have the structuredescribed by (A-V): R₁ is —NHC(═NH)NH₂, R₄ is —CO₂H, and/or R₅ is—COCH₃. In preferred embodiments, A₁ and/or A₂ have the structuredescribed by:

In some embodiments of any of the aspects described herein, A₁ and/or A₂have the structure described by (A-VI):

In preferred embodiments, wherein A₁ and/or A₂ have the structuredescribed by (A-VI): R₁ is —NHC(═NH)NH₂, R₄ is —CO₂H, R₅ is —COCH₃,and/or X is —O—. In preferred embodiments, A₁ and/or A₂ have thestructure of zanamivir described by:

In some embodiments of any of the aspects described herein, A₁ and/or A₂have the structure described by (A-VII):

In preferred embodiments, wherein A₁ and/or A₂ have the structuredescribed by (A-VII): R₁ is —NHC(═NH)NH₂, R₂ is H or F, R₃ is H or F, R₄is —CO₂H, R₅ is —COCH₃, and/or X is —O—. In preferred embodiments, A₁and/or A₂ have the structure described by:

In some embodiments of any of the aspects described herein, A₁ and/or A₂have the structure described by (A-VIII):

In preferred embodiments, wherein A₁ and/or A₂ have the structuredescribed by (A-VIII): R₁ is —NHC(═NH)NH₂, R₅ is —COCH₃, and/or X is—O—. In preferred embodiments, A₁ and/or A₂ have the structure describedby:

In some embodiments of any of the aspects described herein, A₁ and/or A₂have the structure described by (A-IX):

In preferred embodiments, wherein A₁ and/or A₂ have the structuredescribed by (A-IX): R₁ is —NHC(═NH)NH₂, R₂ is H or F, R₃ is H or F, R₅is —COCH₃, and/or X is —O—. In preferred embodiments, A₁ and/or A₂ havethe structure described by:

In some embodiments of any of the aspects described herein, A₁ and/or A₂have the structure described by (A-X):

In preferred embodiments, wherein A₁ and/or A₂ have the structuredescribed by (A-X): R₁ is —NHC(═NH)NH₂, R₃ is H, R₅ is —COCH₃, and/or Xis —O—. In preferred embodiments, A₁ and/or A₂ have the structure ofsulfozanamivir described by:

In some embodiments of any of the aspects described herein, A₁ and/or A₂have the structure described by (A-XI):

In preferred embodiments, wherein A₁ and/or A₂ have the structuredescribed by (A-XI): R₄ is —CO₂H, and/or R₅ is —COCH₃. In preferredembodiments, the alkene is (E), (Z), or a racemic mixture of (E)/(Z). Inpreferred embodiments, A₁ and/or A₂ have the structure of A-315675(Abbott) described by:

In some embodiments of any of the aspects described herein, A₁ and/or A₂have the structure described by (A-XII):

In preferred embodiments, wherein A₁ and/or A₂ have the structuredescribed by (A-XII): R₄ is —CO₂H. In preferred embodiments, A₁ and/orA₂ have the structure of A-315675 (Abbott) described by:

In some embodiments, the conjugate is conjugate 1, or any regioisomerthereof, and the drug-to-antibody ratio (DAR) (e.g., T) is between 0.5and 10.0, e.g., 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5,1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3,3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5,4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9,6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3,7.4, 7.5, 7.6, 7.7, 7.8.0, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7,8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or 10.0. Insome embodiments the DAR is between 0.5 and 2.0, between 2.0 and 4.0,between 4.0 and 6.0 between 6.0 and 8.0, or between 8.0 and 10.0.

In some embodiments, the conjugate is conjugate 2, or any regioisomerthereof, and the DAR (e.g., T) is between 0.5 and 10.0, e.g., 0.5, 0.6,0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2,2.1.2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5,3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0,5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4,6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8.0,7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3,9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or 10.0. In some embodiments the DAR isbetween 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0 between6.0 and 8.0, or between 8.0 and 10.0.

In some embodiments, the conjugate is conjugate 3, or any regioisomerthereof, and the DAR (e.g., T) is between 0.5 and 10.0, e.g., 0.5, 0.6,0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1,2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6,3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1,5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5,6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8.0, 7.9,8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4,9.5, 9.6, 9.7, 9.8, 9.9, or 10.0. In some embodiments the DAR is between0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0 between 6.0 and8.0, or between 8.0 and 10.0.

In some embodiments, the conjugate is conjugate 4, or any regioisomerthereof, and the DAR (e.g., T) is between 0.5 and 10.0, e.g., 0.5, 0.6,0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2,2.1.2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5,3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0,5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4,6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8.0,7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3,9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or 10.0. In some embodiments the DAR isbetween 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0 between6.0 and 8.0, or between 8.0 and 10.0.

In some embodiments, the conjugate is conjugate 5, or any regioisomerthereof, and the DAR (e.g., T) is between 0.5 and 10.0, e.g., 0.5, 0.6,0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2,2.1.2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5,3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0,5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4,6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8.0,7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3,9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or 10.0. In some embodiments the DAR isbetween 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0 between6.0 and 8.0, or between 8.0 and 10.0.

In some embodiments, the conjugate is conjugate 6, or any regioisomerthereof, and the DAR (e.g., T) is between 0.5 and 10.0, e.g., 0.5, 0.6,0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1,2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6,3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1,5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5,6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8.0, 7.9,8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4,9.5, 9.6, 9.7, 9.8, 9.9, or 10.0. In some embodiments the DAR is between0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0 between 6.0 and8.0, or between 8.0 and 10.0.

In some embodiments, the conjugate is conjugate 7, or any regioisomerthereof, and the DAR (e.g., T) is between 0.5 and 10.0, e.g., 0.5, 0.6,0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2,2.1.2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5,3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0,5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4,6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8.0,7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3,9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or 10.0. In some embodiments the DAR isbetween 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0 between6.0 and 8.0, or between 8.0 and 10.0.

In some embodiments, the conjugate is conjugate 8, or any regioisomerthereof, and the DAR (e.g., T) is between 0.5 and 10.0, e.g., 0.5, 0.6,0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2,2.1.2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5,3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0,5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4,6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8.0,7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3,9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or 10.0. In some embodiments the DAR isbetween 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0 between6.0 and 8.0, or between 8.0 and 10.0.

In some embodiments, the conjugate is conjugate 9, or any regioisomerthereof, and the DAR (e.g., T) is between 0.5 and 10.0, e.g., 0.5, 0.6,0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2,2.1.2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5,3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0,5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4,6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8.0,7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3,9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or 10.0. In some embodiments the DAR isbetween 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0 between6.0 and 8.0, or between 8.0 and 10.0.

In some embodiments, the conjugate is conjugate 10, or any regioisomerthereof, and the DAR (e.g., T) is between 0.5 and 10.0, e.g., 0.5, 0.6,0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2,2.1.2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5,3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0,5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4,6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8.0,7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3,9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or 10.0. In some embodiments the DAR isbetween 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0 between6.0 and 8.0, or between 8.0 and 10.0.

In some embodiments, the conjugate is conjugate 11, or any regioisomerthereof, and the DAR (e.g., T) is between 0.5 and 10.0, e.g., 0.5, 0.6,0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1,2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6,3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1,5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5,6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8.0, 7.9,8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4,9.5, 9.6, 9.7, 9.8, 9.9, or 10.0. In some embodiments the DAR is between0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0 between 6.0 and8.0, or between 8.0 and 10.0.

In some embodiments, the conjugate is conjugate 12, or any regioisomerthereof, and the DAR (e.g., T) is between 0.5 and 10.0, e.g., 0.5, 0.6,0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2,2.1.2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5,3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0,5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4,6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8.0,7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3,9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or 10.0. In some embodiments the DAR isbetween 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0 between6.0 and 8.0, or between 8.0 and 10.0.

In some embodiments, the conjugate is conjugate 13, or any regioisomerthereof, and the DAR (e.g., T) is between 0.5 and 10.0, e.g., 0.5, 0.6,0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1,2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6,3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1,5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5,6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8.0, 7.9,8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4,9.5, 9.6, 9.7, 9.8, 9.9, or 10.0. In some embodiments the DAR is between0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0 between 6.0 and8.0, or between 8.0 and 10.0.

In some embodiments, the conjugate is conjugate 14, or any regioisomerthereof, and the DAR (e.g., T) is between 0.5 and 10.0, e.g., 0.5, 0.6,0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2,2.1.2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5,3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0,5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4,6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8.0,7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3,9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or 10.0. In some embodiments the DAR isbetween 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0 between6.0 and 8.0, or between 8.0 and 10.0.

In some embodiments, the conjugate is conjugate 15, or any regioisomerthereof, and the DAR (e.g., T) is between 0.5 and 10.0, e.g., 0.5, 0.6,0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2,2.1.2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5,3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0,5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4,6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8.0,7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3,9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or 10.0. In some embodiments the DAR isbetween 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0 between6.0 and 8.0, or between 8.0 and 10.0.

In some embodiments, the conjugate is conjugate 16, or any regioisomerthereof, and the DAR (e.g., T) is between 0.5 and 10.0, e.g., 0.5, 0.6,0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1,2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6,3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1,5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5,6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8.0, 7.9,8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4,9.5, 9.6, 9.7, 9.8, 9.9, or 10.0. In some embodiments the DAR is between0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0 between 6.0 and8.0, or between 8.0 and 10.0.

In some embodiments, the conjugate is conjugate 17, or any regioisomerthereof, and the DAR (e.g., T) is between 0.5 and 10.0, e.g., 0.5, 0.6,0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2,2.1.2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5,3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0,5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4,6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8.0,7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3,9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or 10.0. In some embodiments the DAR isbetween 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0 between6.0 and 8.0, or between 8.0 and 10.0.

In some embodiments, the conjugate is conjugate 18, or any regioisomerthereof, and the DAR (e.g., T) is between 0.5 and 10.0, e.g., 0.5, 0.6,0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1,2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6,3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1,5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5,6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8.0, 7.9,8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4,9.5, 9.6, 9.7, 9.8, 9.9, or 10.0. In some embodiments the DAR is between0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0 between 6.0 and8.0, or between 8.0 and 10.0.

In some embodiments, the conjugate is conjugate 19, or any regioisomerthereof, and the DAR (e.g., T) is between 0.5 and 10.0, e.g., 0.5, 0.6,0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2,2.1.2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5,3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0,5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4,6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8.0,7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3,9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or 10.0. In some embodiments the DAR isbetween 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0 between6.0 and 8.0, or between 8.0 and 10.0.

In some embodiments, the conjugate is conjugate 20, or any regioisomerthereof, and the DAR (e.g., T) is between 0.5 and 10.0, e.g., 0.5, 0.6,0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2,2.1.2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5,3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0,5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4,6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8.0,7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3,9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or 10.0. In some embodiments the DAR isbetween 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0 between6.0 and 8.0, or between 8.0 and 10.0.

In some embodiments, the conjugate is conjugate 21, or any regioisomerthereof, and the DAR (e.g., T) is between 0.5 and 10.0, e.g., 0.5, 0.6,0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2,2.1.2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5,3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0,5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4,6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8.0,7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3,9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or 10.0. In some embodiments the DAR isbetween 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0 between6.0 and 8.0, or between 8.0 and 10.0.

In some embodiments, the conjugate is conjugate 22, or any regioisomerthereof, and the DAR (e.g., T) is between 0.5 and 10.0, e.g., 0.5, 0.6,0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2,2.1.2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5,3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0,5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4,6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8.0,7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3,9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or 10.0. In some embodiments the DAR isbetween 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0 between6.0 and 8.0, or between 8.0 and 10.0.

In some embodiments, the conjugate is conjugate 23, or any regioisomerthereof, and the DAR (e.g., T) is between 0.5 and 10.0, e.g., 0.5, 0.6,0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1,2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6,3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1,5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5,6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8.0, 7.9,8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4,9.5, 9.6, 9.7, 9.8, 9.9, or 10.0. In some embodiments the DAR is between0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0 between 6.0 and8.0, or between 8.0 and 10.0.

In some embodiments, the conjugate is conjugate 24, or any regioisomerthereof, and the DAR (e.g., T) is between 0.5 and 10.0, e.g., 0.5, 0.6,0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2,2.1.2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5,3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0,5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4,6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8.0,7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3,9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or 10.0. In some embodiments the DAR isbetween 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0 between6.0 and 8.0, or between 8.0 and 10.0.

In some embodiments, the conjugate is conjugate 25, or any regioisomerthereof, and the DAR (e.g., T) is between 0.5 and 10.0, e.g., 0.5, 0.6,0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2,2.1.2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5,3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0,5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4,6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8.0,7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3,9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or 10.0. In some embodiments the DAR isbetween 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0 between6.0 and 8.0, or between 8.0 and 10.0.

In some embodiments, the conjugate is conjugate 26, or any regioisomerthereof, and the DAR (e.g., T) is between 0.5 and 10.0, e.g., 0.5, 0.6,0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2,2.1.2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5,3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0,5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4,6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8.0,7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3,9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or 10.0. In some embodiments the DAR isbetween 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0 between6.0 and 8.0, or between 8.0 and 10.0.

In some embodiments, the conjugate is conjugate 27, or any regioisomerthereof, and the DAR (e.g., T) is between 0.5 and 10.0, e.g., 0.5, 0.6,0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2,2.1.2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5,3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0,5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4,6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8.0,7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3,9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or 10.0. In some embodiments the DAR isbetween 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0 between6.0 and 8.0, or between 8.0 and 10.0.

In some embodiments, the conjugate is conjugate 28, or any regioisomerthereof, and the DAR (e.g., T) is between 0.5 and 10.0, e.g., 0.5, 0.6,0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1,2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6,3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1,5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5,6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8.0, 7.9,8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4,9.5, 9.6, 9.7, 9.8, 9.9, or 10.0. In some embodiments the DAR is between0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0 between 6.0 and8.0, or between 8.0 and 10.0.

In some embodiments, the conjugate is conjugate 29, or any regioisomerthereof, and the DAR (e.g., T) is between 0.5 and 10.0, e.g., 0.5, 0.6,0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2,2.1.2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5,3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0,5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4,6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8.0,7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3,9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or 10.0. In some embodiments the DAR isbetween 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0 between6.0 and 8.0, or between 8.0 and 10.0.

In some embodiments, the conjugate is conjugate 30, or any regioisomerthereof, and the DAR (e.g., T) is between 0.5 and 10.0, e.g., 0.5, 0.6,0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2,2.1.2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5,3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0,5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4,6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8.0,7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3,9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or 10.0. In some embodiments the DAR isbetween 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0 between6.0 and 8.0, or between 8.0 and 10.0.

In some embodiments, the conjugate is conjugate 31, or any regioisomerthereof, and the DAR (e.g., T) is between 0.5 and 10.0, e.g., 0.5, 0.6,0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2,2.1.2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5,3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0,5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4,6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8.0,7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3,9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or 10.0. In some embodiments the DAR isbetween 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0 between6.0 and 8.0, or between 8.0 and 10.0.

In some embodiments, the conjugate is conjugate 32, or any regioisomerthereof, and the DAR (e.g., T) is between 0.5 and 10.0, e.g., 0.5, 0.6,0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2,2.1.2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5,3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0,5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4,6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8.0,7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3,9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or 10.0. In some embodiments the DAR isbetween 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0 between6.0 and 8.0, or between 8.0 and 10.0.

In some embodiments, the conjugate is conjugate 33, or any regioisomerthereof, and the DAR (e.g., T) is between 0.5 and 10.0, e.g., 0.5, 0.6,0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1,2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6,3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1,5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5,6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8.0, 7.9,8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4,9.5, 9.6, 9.7, 9.8, 9.9, or 10.0. In some embodiments the DAR is between0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0 between 6.0 and8.0, or between 8.0 and 10.0.

In some embodiments, the conjugate is conjugate 34, or any regioisomerthereof, and the DAR (e.g., T) is between 0.5 and 10.0, e.g., 0.5, 0.6,0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2,2.1.2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5,3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0,5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4,6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8.0,7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3,9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or 10.0. In some embodiments the DAR isbetween 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0 between6.0 and 8.0, or between 8.0 and 10.0.

In some embodiments, the conjugate is conjugate 35, or any regioisomerthereof, and the DAR (e.g., T) is between 0.5 and 10.0, e.g., 0.5, 0.6,0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1,2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6,3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1,5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5,6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8.0, 7.9,8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4,9.5, 9.6, 9.7, 9.8, 9.9, or 10.0. In some embodiments the DAR is between0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0 between 6.0 and8.0, or between 8.0 and 10.0.

In some embodiments, the conjugate is conjugate 36, or any regioisomerthereof, and the DAR (e.g., T) is between 0.5 and 10.0, e.g., 0.5, 0.6,0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2,2.1.2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5,3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0,5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4,6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8.0,7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3,9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or 10.0. In some embodiments the DAR isbetween 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0 between6.0 and 8.0, or between 8.0 and 10.0.

In some embodiments, the conjugate is conjugate 37, or any regioisomerthereof, and the DAR (e.g., T) is between 0.5 and 10.0, e.g., 0.5, 0.6,0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2,2.1.2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5,3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0,5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4,6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8.0,7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3,9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or 10.0. In some embodiments the DAR isbetween 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0 between6.0 and 8.0, or between 8.0 and 10.0.

In some embodiments, the conjugate is conjugate 38, or any regioisomerthereof, and the DAR (e.g., T) is between 0.5 and 10.0, e.g., 0.5, 0.6,0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2,2.1.2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5,3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0,5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4,6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8.0,7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3,9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or 10.0. In some embodiments the DAR isbetween 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0 between6.0 and 8.0, or between 8.0 and 10.0.

In some embodiments, the conjugate is conjugate 39, or any regioisomerthereof, and the DAR (e.g., T) is between 0.5 and 10.0, e.g., 0.5, 0.6,0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2,2.1.2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5,3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0,5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4,6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8.0,7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3,9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or 10.0. In some embodiments the DAR isbetween 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0 between6.0 and 8.0, or between 8.0 and 10.0.

In some embodiments, the conjugate is conjugate 40, or any regioisomerthereof, and the DAR (e.g., T) is between 0.5 and 10.0, e.g., 0.5, 0.6,0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1,2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6,3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1,5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5,6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8.0, 7.9,8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4,9.5, 9.6, 9.7, 9.8, 9.9, or 10.0. In some embodiments the DAR is between0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0 between 6.0 and8.0, or between 8.0 and 10.0.

In some embodiments, the conjugate is conjugate 41, or any regioisomerthereof, and the DAR (e.g., T) is between 0.5 and 10.0, e.g., 0.5, 0.6,0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2,2.1.2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5,3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0,5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4,6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8.0,7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3,9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or 10.0. In some embodiments the DAR isbetween 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0 between6.0 and 8.0, or between 8.0 and 10.0.

In some embodiments, the conjugate is conjugate 42, or any regioisomerthereof, and the DAR (e.g., T) is between 0.5 and 10.0, e.g., 0.5, 0.6,0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2,2.1.2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5,3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0,5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4,6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8.0,7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3,9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or 10.0. In some embodiments the DAR isbetween 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0 between6.0 and 8.0, or between 8.0 and 10.0.

In some embodiments, the conjugate is conjugate 43, or any regioisomerthereof, and the DAR (e.g., T) is between 0.5 and 10.0, e.g., 0.5, 0.6,0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2,2.1.2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5,3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0,5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4,6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8.0,7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3,9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or 10.0. In some embodiments the DAR isbetween 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0 between6.0 and 8.0, or between 8.0 and 10.0.

Definitions

To facilitate the understanding of this invention, a number of terms aredefined below. Terms defined herein have meanings as commonly understoodby a person of ordinary skill in the areas relevant to the presentinvention. Terms such as “a”, “an,” and “the” are not intended to referto only a singular entity, but include the general class of which aspecific example may be used for illustration. The terminology herein isused to describe specific embodiments of the invention, but their usagedoes not delimit the invention, except as outlined in the claims.

The term “neuraminidase inhibitor” or “viral neuraminidase inhibitor,”as used herein, refers to compounds that decreases the activity of theenzyme influenza virus neuraminidase (e.g., from influenza virus A, B,or C). A neuraminidase inhibitor may be identified by methods known tothose of skill in the art, for example, by reduction of viralreplication in an influenza viral plaque reduction assay, e.g., atconcentrations less than 20 μM (e.g., less than 10 μM, 5 μM, 2 μM, 1 μM,500 nM or 100 nM). Viral neuraminidase inhibitors known to those ofskill in the art include zanamivir, sulfozanamivir, peramivir, andA-315675 (Abbott) (see, for example, Hadházi et al. A sulfozanamiviranalogue has potent anti-influenza virus activity. ChemMedChem Comm.13:785-789 (2018) and In vitro characterization of A-315675, a highlypotent inhibitor of A and B strain of influenze virus neuraminidases andinfluenza virus replication. Antimicrobial Agents and Chemotherapy46(4):1014-1021 (2002)). Viral neuraminidase inhibitors of the inventioninclude zanamivir, sulfozanamivir, peramavir, A-315675 and analogsthereof, such as the viral neuraminidase inhibitors of formulas(A-I)-(A-XII):

wherein R₁ is selected from —OH, —NH₂, —NHC(═NH)NH₂, and —NHC(═NH)NHR₆;R₂ and R₃ are each independently selected from —H, —OH, —F, —Cl, and—Br; R₄ is selected from —CO₂H, —P(═O)(OH)₂, —SO₃H; R₅ is selected from—COCH₃, —COCF₃, —SO₂CH₃; X is selected from —O— and —S—; Y is selectedfrom

R₆ is selected from

R₇ is selected from H, C1-C20 alkyl, C3-C20 cycloalkyl, C3-C20heterocycloalkyl; C5-C15 aryl, and C2-C15 heteroaryl; and R₈ is selectedfrom C3-C20 heterocycloalkyl, C5-C15 aryl, and C2-C15 heteroaryl. Theterm “inhibits neuraminidase activity,” as used herein refers to an IC₅₀of less than or equal to 1,000 nM, for example, as measured inaccordance with the neuraminidase inhibition assay in Example 2 herein.Specifically, the IC₅₀ represents the concentration of the influenzavirus neuraminidase inhibitor that is required for 50% inhibition invitro. In some aspects, an IC₅₀ of less than or equal to 100 nM or lessthan or equal to 10 nM in accordance with neuraminidase inhibition assayis indicative of a compound inhibiting neuraminidase activity.

By “viral infection” is meant the pathogenic growth of a virus (e.g.,the influenza virus) in a host organism (e.g., a human subject). A viralinfection can be any situation in which the presence of a viralpopulation(s) is damaging to a host body. Thus, a subject is “suffering”from a viral infection when an excessive amount of a viral population ispresent in or on the subject's body, or when the presence of a viralpopulation(s) is damaging the cells or other tissue of the subject.

As used herein, the term “Fc domain monomer” refers to a polypeptidechain that includes at least a hinge domain and second and thirdantibody constant domains (C_(H)2 and C_(H)3) or functional fragmentsthereof (e.g., fragments that that capable of (i) dimerizing withanother Fc domain monomer to form an Fc domain, and (ii) binding to anFc receptor. The Fc domain monomer can be any immunoglobulin antibodyisotype, including IgG, IgE, IgM, IgA, or IgD (e.g., IgG). Additionally,the Fc domain monomer can be an IgG subtype (e.g., IgG1, IgG2a, IgG2b,IgG3, or IgG4) (e.g., IgG1). An Fc domain monomer does not include anyportion of an immunoglobulin that is capable of acting as anantigen-recognition region, e.g., a variable domain or a complementaritydetermining region (CDR). Fc domain monomers in the conjugates asdescribed herein can contain one or more changes from a wild-type Fcdomain monomer sequence (e.g., 1-10, 1-8, 1-6, 1-4 amino acidsubstitutions, additions, or deletions) that alter the interactionbetween an Fc domain and an Fc receptor. Examples of suitable changesare known in the art. In certain embodiments, a human Fc domain monomer(e.g., an IgG heavy chain, such as IgG1) comprises a region that extendsfrom any of Asn208, Glu216, Asp221, Lys222, or Cys226 to thecarboxyl-terminus of the heavy chain at Lys447. C-terminal Lys447 of theFc region may or may not be present, without affecting the structure orstability of the Fc region. Unless otherwise specified herein, numberingof amino acid residues in the IgG or Fc domain monomer is according tothe EU numbering system for antibodies, also called the Kabat EU index,as described, for example, in Kabat et al., Sequences of Proteins ofImmunological Interest, 5th Ed. Public Health Service, NationalInstitutes of Health, Bethesda, Md., 1991.

As used herein, the term “Fc domain” refers to a dimer of two Fc domainmonomers that is capable of binding an Fc receptor. In the wild-type Fcdomain, the two Fc domain monomers dimerize by the interaction betweenthe two C_(H)3 antibody constant domains, in some embodiments, one ormore disulfide bonds form between the hinge domains of the twodimerizing Fc domain monomers. The term “covalently attached” refers totwo parts of a conjugate that are linked to each other by a covalentbond formed between two atoms in the two parts of the conjugate.

As used herein, the term “Fc-binding peptide” refers to refers to apolypeptide having an amino acid sequence of 5 to 50 (e.g., 5 to 40, 5to 30, 5 to 20, 5 to 15, 5 to 10, 10 to 50, 10 to 30, or 10 to 20) aminoacid residues that has affinity for and functions to bind an Fc domain,such as any of the Fc domain described herein. An Fc-binding peptidepeptide can be of different origins, e.g., synthetic, human, mouse, orrat. Fc-binding peptides of the invention include Fc-binding peptideswhich have been engineered to include one or more (e.g., two, three,four, or five) solvent-exposed cysteine or lysine residues, which mayprovide a site for conjugation to a compound of the invention (e.g.,conjugation to a neuraminidase inhibitor monomer or dimer, including byway of a linker). Most preferably, the Fc-binding peptide will contain asingle solvent-exposed cysteine or lysine, thus enabling site-specificconjugation of a compound of the invention. Fc-binding peptides mayinclude only naturally occurring amino acid residues, or may include oneor more non-naturally occurring amino acid residues. Where included, anon-naturally occurring amino acid residue (e.g., the side chain of anon-naturally occurring amino acid residue) may used as the point ofattachment for a compound of the invention (e.g., a neuraminidaseinhibitor monomer or dimer, including by way of a linker). Fc-bindingpeptides of the invention may be linear or cyclic. Fc-binding peptidesof the invention include any Fc-binding peptides known to one of skillin the art.

As used here, the term “albumin protein” refers to a polypeptidecomprising an amino acid sequence corresponding to a naturally-occurringalbumin protein (e.g., human serum albumin) or a variant thereof, suchas an engineered variant of a naturally-occurring albumin protein.Variants of albumin proteins include polymorphisms, fragments such asdomains and sub-domains, and fusion proteins (e.g., an albumin proteinhaving a C-terminal or N-terminal fusion, such as a polypeptide linker).Preferably the albumin protein has the amino acid sequence of humanserum albumin (HSA) or a variant or fragment thereof, most preferably afunctional variant or fragment thereof. Albumin proteins of theinvention include proteins having at least 70%, 75%, 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to any one of SEQ IDNOs: 69-71. Albumin proteins of the invention include albumin proteinswhich have been engineered to include one or more (e.g., two, three,four, or five) solvent-exposed cysteine or lysine residues, which mayprovide a site for conjugation to a compound of the invention (e.g.,conjugation to a neuraminidase inhibitor monomer or dimer, including byway of a linker). Most preferably, the albumin protein will contain asingle solvent-exposed cysteine or lysine, thus enabling site-specificconjugation of a compound of the invention. Albumin proteins may includeonly naturally occurring amino acid residues, or may include one or morenon-naturally occurring amino acid residues. Where included, anon-naturally occurring amino acid residue (e.g., the side chain of anon-naturally occurring amino acid residue) may used as the point ofattachment for a compound of the invention (e.g., a neuraminidaseinhibitor monomer or dimer, including byway of a linker).

As used herein, the term “albumin protein-binding peptide” refers to apolypeptide having an amino acid sequence of 5 to 50 (e.g., 5 to 40, 5to 30, 5 to 20, 5 to 15, 5 to 10, 10 to 50, 10 to 30, or 10 to 20) aminoacid residues that has affinity for and functions to bind an albuminprotein, such as any of the albumin proteins described herein.Preferably, the albumin protein-binding peptide binds to anaturally-occurring serum albumin, most preferably human serum albumin.An albumin protein-binding peptide can be of different origins, e.g.,synthetic, human, mouse, or rat. Albumin protein-binding peptides of theinvention include albumin protein-binding peptides which have beenengineered to include one or more (e.g., two, three, four, or five)solvent-exposed cysteine or lysine residues, which may provide a sitefor conjugation to a compound of the invention (e.g., conjugation to aneuraminidase inhibitor monomer or dimer, including by way of a linker).Most preferably, the albumin protein-binding peptide will contain asingle solvent-exposed cysteine or lysine, thus enabling site-specificconjugation of a compound of the invention. Albumin protein-bindingpeptides may include only naturally occurring amino acid residues, ormay include one or more non-naturally occurring amino acid residues.Where included, a non-naturally occurring amino acid residue (e.g., theside chain of a non-naturally occurring amino acid residue) may be usedas the point of attachment for a compound of the invention (e.g., aneuraminidase inhibitor monomer or dimer, including by way of a linker).Albumin protein-binding peptides of the invention may be linear orcyclic. Albumin protein-binding peptide of the invention include anyalbumin protein-binding peptides known to one of skill in the art,examples of which, are provided herein. Further exemplary albuminprotein-binding peptides are provided in U.S. Patent Application No.2005/0287153, which is incorporated herein by reference in its entirety.

As used-herein, a “surface exposed amino acid” or “solvent-exposed aminoacid,” such as a surface exposed cysteine or a surface exposed lysinerefers to an amino acid that is accessible to the solvent surroundingthe protein. A surface exposed amino acid may be a naturally-occurringor an engineered variant (e.g., a substitution or insertion) of theprotein. In some embodiments, a surface exposed amino acid is an aminoacid that when substituted does not substantially change thethree-dimensional structure of the protein.

The terms “linker,” “L,” and “L′,” as used herein, refer to a covalentlinkage or connection between two or more components in a conjugate(e.g., between two neuraminidase inhibitors in a conjugate describedherein, between a neuraminidase inhibitor and an Fc domain or albuminprotein in a conjugate described herein, and between a dimer of twoneuraminidase inhibitors and an Fc domain or an albumin protein in aconjugate described herein). In some embodiments, a conjugate describedherein may contain a linker that has a trivalent structure (e.g., atrivalent linker). A trivalent linker has three arms, in which each armis covalently linked to a component of the conjugate (e.g., a first armconjugated to a first neuraminidase inhibitor, a second arm conjugatedto a second neuraminidase inhibitor, and a third arm conjugated to an Fcdomain or an albumin protein).

Molecules that may be used as linkers include at least two functionalgroups, which may be the same or different, e.g., two carboxylic acidgroups, two amine groups, two sulfonic acid groups, a carboxylic acidgroup and a maleimide group, a carboxylic acid group and an alkynegroup, a carboxylic acid group and an amine group, a carboxylic acidgroup and a sulfonic acid group, an amine group and a maleimide group,an amine group and an alkyne group, or an amine group and a sulfonicacid group. The first functional group may form a covalent linkage witha first component in the conjugate and the second functional group mayform a covalent linkage with the second component in the conjugate. Insome embodiments of a trivalent linker, two arms of a linker may containtwo dicarboxylic acids, in which the first carboxylic acid may form acovalent linkage with the first neuraminidase inhibitor in the conjugateand the second carboxylic acid may form a covalent linkage with thesecond neuraminidase inhibitor in the conjugate, and the third arm ofthe linker may for a covalent linkage with an Fc domain or albuminprotein in the conjugate. Examples of dicarboxylic acids are describedfurther herein. In some embodiments, a molecule containing one or moremaleimide groups may be used as a linker, in which the maleimide groupmay form a carbon-sulfur linkage with a cysteine in a component (e.g.,an Fc domain or an albumin protein) in the conjugate. In someembodiments, a molecule containing one or more alkyne groups may be usedas a linker, in which the alkyne group may form a 1,2,3-triazole linkagewith an azide in a component (e.g., an Fc domain or an albumin protein)in the conjugate. In some embodiments, a molecule containing one or moreazide groups may be used as a linker, in which the azide group may forma 1,2,3-triazole linkage with an alkyne in a component (e.g., an Fcdomain or an albumin protein) in the conjugate. In some embodiments, amolecule containing one or more bis-sulfone groups may be used as alinker, in which the bis-sulfone group may form a linkage with an aminegroup a component (e.g., an Fc domain or an albumin protein) in theconjugate. In some embodiments, a molecule containing one or moresulfonic acid groups may be used as a linker, in which the sulfonic acidgroup may form a sulfonamide linkage with a component in the conjugate.In some embodiments, a molecule containing one or more isocyanate groupsmay be used as a linker, in which the isocyanate group may form a urealinkage with a component in the conjugate. In some embodiments, amolecule containing one or more haloalkyl groups may be used as alinker, in which the haloalkyl group may form a covalent linkage, e.g.,C—N and C—O linkages, with a component in the conjugate.

In some embodiments, a linker provides space, rigidity, and/orflexibility between the two or more components. In some embodiments, alinker may be a bond, e.g., a covalent bond. The term “bond” refers to achemical bond, e.g., an amide bond, a disulfide bond, a C—O bond, a C—Nbond, a N—N bond, a C—S bond, or any kind of bond created from achemical reaction, e.g., chemical conjugation. In some embodiments, alinker includes no more than 250 atoms. In some embodiments, a linkerincludes no more than 250 non-hydrogen atoms. In some embodiments, thebackbone of a linker includes no more than 250 atoms. The “backbone” ofa linker refers to the atoms in the linker that together form theshortest path from one part of a conjugate to another part of theconjugate (e.g., the shortest path linking a first neuraminidaseinhibitor and a second neuraminidase inhibitor). The atoms in thebackbone of the linker are directly involved in linking one part of aconjugate to another part of the conjugate (e.g., linking a firstneuraminidase inhibitor and a second neuraminidase inhibitor). Forexamples, hydrogen atoms attached to carbons in the backbone of thelinker are not considered as directly involved in linking one part ofthe conjugate to another part of the conjugate.

In some embodiments, a linker may comprise a synthetic group derivedfrom, e.g., a synthetic polymer (e.g., a polyethylene glycol (PEG)polymer). In some embodiments, a linker may comprise one or more aminoacid residues, such as D- or L-amino acid residues. In some embodiments,a linker may be a residue of an amino acid sequence (e.g., a 1-25 aminoacid, 1-10 amino acid, 1-9 amino acid, 1-8 amino acid, 1-7 amino acid,1-6 amino acid, 1-5 amino acid, 1-4 amino acid, 1-3 amino acid, 1-2amino acid, or 1 amino acid sequence). In some embodiments, a linker maycomprise one or more, e.g., 1-100, 1-50, 1-25, 1-10, 1-5, or 1-3,optionally substituted alkylene, optionally substituted heteroalkylene(e.g., a PEG unit), optionally substituted alkenylene, optionallysubstituted heteroalkenylene, optionally substituted alkynylene,optionally substituted heteroalkynylene, optionally substitutedcycloalkylene, optionally substituted heterocycloalkylene, optionallysubstituted cycloalkenylene, optionally substitutedheterocycloalkenylene, optionally substituted cycloalkynylene,optionally substituted heterocycloalkynylene, optionally substitutedarylene, optionally substituted heteroarylene (e.g., pyridine), O, S,NR^(i) (R^(i) is H, optionally substituted alkyl, optionally substitutedheteroalkyl, optionally substituted alkenyl, optionally substitutedheteroalkenyl, optionally substituted alkynyl, optionally substitutedheteroalkynyl, optionally substituted cycloalkyl, optionally substitutedheterocycloalkyl, optionally substituted cycloalkenyl, optionallysubstituted heterocycloalkenyl, optionally substituted cycloalkynyl,optionally substituted heterocycloalkynyl, optionally substituted aryl,or optionally substituted heteroaryl), P, carbonyl, thiocarbonyl,sulfonyl, phosphate, phosphoryl, or imino. For example, a linker maycomprise one or more optionally substituted C1-C20 alkylene, optionallysubstituted C1-C20 heteroalkylene (e.g., a PEG unit), optionallysubstituted C2-C20 alkenylene (e.g., C2 alkenylene), optionallysubstituted C2-C20 heteroalkenylene, optionally substituted C2-C20alkynylene, optionally substituted C2-C20 heteroalkynylene, optionallysubstituted C3-C20 cycloalkylene (e.g., cyclopropylene, cyclobutylene),optionally substituted C3-C20 heterocycloalkylene, optionallysubstituted C4-C20 cycloalkenylene, optionally substituted C4-C20heterocycloalkenylene, optionally substituted C8-C20 cycloalkynylene,optionally substituted C8-C20 heterocycloalkynylene, optionallysubstituted C5-C15 arylene (e.g., C6 arylene), optionally substitutedC2-C15 heteroarylene (e.g., imidazole, pyridine), O, S, NR^(i) (R^(i) isH, optionally substituted C1-C20 alkyl, optionally substituted C1-C20heteroalkyl, optionally substituted C2-C20 alkenyl, optionallysubstituted C2-C20 heteroalkenyl, optionally substituted C2-C20 alkynyl,optionally substituted C2-C20 heteroalkynyl, optionally substitutedC3-C20 cycloalkyl, optionally substituted C3-C20 heterocycloalkyl,optionally substituted C4-C20 cycloalkenyl, optionally substitutedC4-C20 heterocycloalkenyl, optionally substituted C8-C20 cycloalkynyl,optionally substituted C8-C20 heterocycloalkynyl, optionally substitutedC5-C15 aryl, or optionally substituted C2-C15 heteroaryl), P, carbonyl,thiocarbonyl, sulfonyl, phosphate, phosphoryl, or imino.

The terms “alkyl,” “alkenyl,” and “alkynyl,” as used herein, includestraight-chain and branched-chain monovalent substituents, as well ascombinations of these, containing only C and H when unsubstituted. Whenthe alkyl group includes at least one carbon-carbon double bond orcarbon-carbon triple bond, the alkyl group can be referred to as an“alkenyl” or “alkynyl” group respectively. The monovalency of an alkyl,alkenyl, or alkynyl group does not include the optional substituents onthe alkyl, alkenyl, or alkynyl group. For example, if an alkyl, alkenyl,or alkynyl group is attached to a compound, monovalency of the alkyl,alkenyl, or alkynyl group refers to its attachment to the compound anddoes not include any additional substituents that may be present on thealkyl, alkenyl, or alkynyl group. In some embodiments, the alkyl orheteroalkyl group may contain, e.g., 1-20, 1-18, 1-16, 1-14, 1-12, 1-10,1-8, 1-6, 1-4, or 1-2 carbon atoms (e.g., C1-C20, C1-C18, C1-C16,C1-C14, C1-C12, C1-C10, C1-C8, C1-C6, C1-C4, or C1-C2). In someembodiments, the alkenyl, heteroalkenyl, alkynyl, or heteroalkynyl groupmay contain, e.g., 2-20, 2-18, 2-16, 2-14, 2-12, 2-10, 2-8, 2-6, or 2-4carbon atoms (e.g., C2-C20, C2-C18, C2-C16, C2-C14, C2-C12, C2-C10,C2-C8, C2-C6, or C2-C4). Examples include, but are not limited to,methyl, ethyl, isobutyl, sec-butyl, tert-butyl, 2-propenyl, and3-butynyl.

The term “cycloalkyl,” as used herein, represents a monovalent saturatedor unsaturated non-aromatic cyclic alkyl group. A cycloalkyl may have,e.g., three to twenty carbons (e.g., a C3-C7, C3-C8, C3-C9, C3-C10,C3-C11, C3-C12, C3-C14, C3-C16, C3-C18, or C3-C20 cycloalkyl). Examplesof cycloalkyls include, but are not limited to, cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl, and cycloheptyl. When the cycloalkyl groupincludes at least one carbon-carbon double bond, the cycloalkyl groupcan be referred to as a “cycloalkenyl” group. A cycloalkenyl may have,e.g., four to twenty carbons (e.g., a C4-C7, C4-C8, C4-C9, C4-C10,C4-C11, C4-C12, C4-C14, C4-C16, C4-C18, or C4-C20 cycloalkenyl).Exemplary cycloalkenyl groups include, but are not limited to,cyclopentenyl, cyclohexenyl, and cycloheptenyl. When the cycloalkylgroup includes at least one carbon-carbon triple bond, the cycloalkylgroup can be referred to as a “cycloalkynyl” group. A cycloalkynyl mayhave, e.g., eight to twenty carbons (e.g., a C8-C9, C8-C10, C8-C11,C8-C12, C8-C14, C8-C16, C8-C18, or C8-C20 cycloalkynyl). The term“cycloalkyl” also includes a cyclic compound having a bridgedmulticyclic structure in which one or more carbons bridges twonon-adjacent members of a monocyclic ring, e.g., bicyclo[2.2.1.]heptyland adamantane. The term “cycloalkyl” also includes bicyclic, tricyclic,and tetracyclic fused ring structures, e.g., decalin and spiro cycliccompounds.

The term “aryl,” as used herein, refers to any monocyclic or fused ringbicyclic or tricyclic system which has the characteristics ofaromaticity in terms of electron distribution throughout the ringsystem, e.g., phenyl, naphthyl, or phenanthrene. In some embodiments, aring system contains 5-15 ring member atoms or 5-10 ring member atoms.An aryl group may have, e.g., five to fifteen carbons (e.g., a C5-C6,C5-C7, C5-C8, C5-C9, C5-C10, C5-C11, C5-C12, C5-C13, C5-C14, or C5-C15aryl). The term “heteroaryl” also refers to such monocyclic or fusedbicyclic ring systems containing one or more, e.g., 1-4, 1-3, 1, 2, 3,or 4, heteroatoms selected from O, S and N. A heteroaryl group may have,e.g., two to fifteen carbons (e.g., a C2-C3, C2-C4, C2-C5, C2-C6, C2-C7,C2-C8, C2-C9. C2-C10, C2-C11, C2-C12, C2-C13, C2-C14, or C2-C15heteroaryl). The inclusion of a heteroatom permits inclusion of5-membered rings to be considered aromatic as well as 6-membered rings.Thus, typical heteroaryl systems include, e.g., pyridyl, pyrimidyl,indolyl, benzimidazolyl, benzotriazolyl, isoquinolyl, quinolyl,benzothiazolyl, benzofuranyl, thienyl, furyl, pyrrolyl, thiazolyl,oxazolyl, isoxazolyl, benzoxazolyl, benzoisoxazolyl, and imidazolyl.Because tautomers are possible, a group such as phthalimido is alsoconsidered heteroaryl. In some embodiments, the aryl or heteroaryl groupis a 5- or 6-membered aromatic rings system optionally containing 1-2nitrogen atoms. In some embodiments, the aryl or heteroaryl group is anoptionally substituted phenyl, pyridyl, indolyl, pyrimidyl, pyridazinyl,benzothiazolyl, benzimidazolyl, pyrazolyl, imidazolyl, isoxazolyl,thiazolyl, or imidazopyridinyl. In some embodiments, the aryl group isphenyl. In some embodiments, an aryl group may be optionally substitutedwith a substituent such an aryl substituent, e.g., biphenyl.

The term “alkaryl,” refers to an aryl group that is connected to analkylene, alkenylene, or alkynylene group. In general, if a compound isattached to an alkaryl group, the alkylene, alkenylene, or alkynyleneportion of the alkaryl is attached to the compound. In some embodiments,an alkaryl is C6-C35 alkaryl (e.g., C6-C16, C6-C14, C6-C12, C6-C10,C6-C9, C6-C8, C7, or C6 alkaryl), in which the number of carbonsindicates the total number of carbons in both the aryl portion and thealkylene, alkenylene, or alkynylene portion of the alkaryl. Examples ofalkaryls include, but are not limited to, (C1-C8)alkylene(C6-C12)aryl,(C2-C8)alkenylene(C6-C12)aryl, or (C2-C8)alkynylene(C6-C12)aryl. In someembodiments, an alkaryl is benzyl or phenethyl. In a heteroalkaryl, oneor more heteroatoms selected from N, O, and S may be present in thealkylene, alkenylene, or alkynylene portion of the alkaryl group and/ormay be present in the aryl portion of the alkaryl group. In anoptionally substituted alkaryl, the substituent may be present on thealkylene, alkenylene, or alkynylene portion of the alkaryl group and/ormay be present on the aryl portion of the alkaryl group.

The term “amino,” as used herein, represents —N(R^(x))₂ or —N⁺(R^(x))₃,where each R^(x) is, independently, H, alkyl, alkenyl, alkynyl, aryl,alkaryl, cycloalkyl, or two R^(x) combine to form a heterocycloalkyl. Insome embodiment, the amino group is —NH₂.

The term “alkamino,” as used herein, refers to an amino group, describedherein, that is attached to an alkylene (e.g., C1-C5 alkylene),alkenylene (e.g., C2-C5 alkenylene), or alkynylene group (e.g., C2-C5alkenylene). In general, if a compound is attached to an alkamino group,the alkylene, alkenylene, or alkynylene portion of the alkamino isattached to the compound. The amino portion of an alkamino refers to—N(R^(x))₂ or —N⁺(R^(x))₃, where each R^(x) is, independently, H, alkyl,alkenyl, alkynyl, aryl, alkaryl, cycloalkyl, or two R^(x) combine toform a heterocycloalkyl. In some embodiment, the amino portion of analkamino is —NH₂. An example of an alkamino group is C1-C5 alkamino,e.g., C2 alkamino (e.g., CH₂CH₂NH₂ or CH₂CH₂N(CH₃)₂). In aheteroalkamino group, one or more, e.g., 1-4, 1-3, 1, 2, 3, or 4,heteroatoms selected from N, O, and S may be present in the alkylene,alkenylene, or alkynylene portion of the heteroalkamino group. In someembodiments, an alkamino group may be optionally substituted. In asubstituted alkamino group, the substituent may be present on thealkylene, alkenylene, or alkynylene portion of the alkamino group and/ormay be present on the amino portion of the alkamino group.

The term “alkamide,” as used herein, refers to an amide group that isattached to an alkylene (e.g., C1-C5 alkylene), alkenylene (e.g., C2-C5alkenylene), or alkynylene (e.g., C2-C5 alkenylene) group. In general,if a compound is attached to an alkamide group, the alkylene,alkenylene, or alkynylene portion of the alkamide is attached to thecompound. The amide portion of an alkamide refers to —C(O)—N(R^(x))₂,where each R^(x) is, independently, H, alkyl, alkenyl, alkynyl, aryl,alkaryl, cycloalkyl, or two R^(x) combine to form a heterocycloalkyl. Insome embodiment, the amide portion of an alkamide is —C(O)NH₂. Analkamide group may be —(CH₂)₂—C(O)NH₂ or —CH₂—C(O)NH₂. In aheteroalkamide group, one or more, e.g., 1-4, 1-3, 1, 2, 3, or 4,heteroatoms selected from N, O, and S may be present in the alkylene,alkenylene, or alkynylene portion of the heteroalkamide group. In someembodiments, an alkamide group may be optionally substituted. In asubstituted alkamide group, the substituent may be present on thealkylene, alkenylene, or alkynylene portion of the alkamide group and/ormay be present on the amide portion of the alkamide group.

The terms “alkylene,” “alkenylene,” and “alkynylene,” as used herein,refer to divalent groups having a specified size. In some embodiments,an alkylene may contain, e.g., 1-20, 1-18, 1-16, 1-14, 1-12, 1-10, 1-8,1-6, 1-4, or 1-2 carbon atoms (e.g., C1-C20, C1-C18, C1-C16, C1-C14,C1-C12, C1-C10, C1-C8, C1-C6, C1-C4, or C1-C2). In some embodiments, analkenylene or alkynylene may contain, e.g., 2-20, 2-18, 2-16, 2-14,2-12, 2-10, 2-8, 2-6, or 2-4 carbon atoms (e.g., C2-C20, C2-C18, C2-C16,C2-C14, C2-C12, C2-C10, C2-C8, C2-C6, or C2-C4). Alkylene, alkenylene,and/or alkynylene includes straight-chain and branched-chain forms, aswell as combinations of these. The divalency of an alkylene, alkenylene,or alkynylene group does not include the optional substituents on thealkylene, alkenylene, or alkynylene group. For example, twoneuraminidase inhibitors may be attached to each other by way of alinker that includes alkylene, alkenylene, and/or alkynylene, orcombinations thereof. Each of the alkylene, alkenylene, and/oralkynylene groups in the linker is considered divalent with respect tothe two attachments on either end of alkylene, alkenylene, and/oralkynylene group. For example, if a linker includes -(optionallysubstituted alkylene)-(optionally substituted alkenylene)-(optionallysubstituted alkylene)-, the alkenylene is considered divalent withrespect to its attachments to the two alkylenes at the ends of thelinker. The optional substituents on the alkenylene are not included inthe divalency of the alkenylene. The divalent nature of an alkylene,alkenylene, or alkynylene group (e.g., an alkylene, alkenylene, oralkynylene group in a linker) refers to both of the ends of the groupand does not include optional substituents that may be present in analkylene, alkenylene, or alkynylene group. Because they are divalent,they can link together multiple (e.g., two) parts of a conjugate, e.g.,a first neuraminidase inhibitor and a second neuraminidase inhibitor.Alkylene, alkenylene, and/or alkynylene groups can be substituted by thegroups typically suitable as substituents for alkyl, alkenyl and alkynylgroups as set forth herein. For example, C═O is a C1 alkylene that issubstituted by an oxo (═O). For example, —HCR—C≡C— may be considered asan optionally substituted alkynylene and is considered a divalent groupeven though it has an optional substituent, R. Heteroalkylene,heteroalkenylene, and/or heteroalkynylene groups refer to alkylene,alkenylene, and/or alkynylene groups including one or more, e.g., 1-4,1-3, 1, 2, 3, or 4, heteroatoms, e.g., N, O, and S. For example, apolyethylene glycol (PEG) polymer or a PEG unit —(CH₂)₂—O— in a PEGpolymer is considered a heteroalkylene containing one or more oxygenatoms.

The term “cycloalkylene,” as used herein, refers to a divalent cyclicgroup linking together two parts of a compound. For example, one carbonwithin the cycloalkylene group may be linked to one part of thecompound, while another carbon within the cycloalkylene group may belinked to another part of the compound. A cycloalkylene group mayinclude saturated or unsaturated non-aromatic cyclic groups. Acycloalkylene may have, e.g., three to twenty carbons in the cyclicportion of the cycloalkylene (e.g., a C3-C7, C3-C8, C3-C9, C3-C10,C3-C11, C3-C12, C3-C14, C3-C16, C3-C18, or C3-C20 cycloalkylene). Whenthe cycloalkylene group includes at least one carbon-carbon double bond,the cycloalkylene group can be referred to as a “cycloalkenylene” group.A cycloalkenylene may have, e.g., four to twenty carbons in the cyclicportion of the cycloalkenylene (e.g., a C4-C7, C4-C8, C4-C9. C4-C10,C4-C11, C4-C12, C4-C14, C4-C16, C4-C18, or C4-C20 cycloalkenylene). Whenthe cycloalkylene group includes at least one carbon-carbon triple bond,the cycloalkylene group can be referred to as a “cycloalkynylene” group.A cycloalkynylene may have, e.g., four to twenty carbons in the cyclicportion of the cycloalkynylene (e.g., a C4-C7, C4-C8, C4-C9. C4-C10,C4-C11, C4-C12, C4-C14, C4-C16, C4-C18, or C8-C20 cycloalkynylene). Acycloalkylene group can be substituted by the groups typically suitableas substituents for alkyl, alkenyl and alkynyl groups as set forthherein. Heterocycloalkylene refers to a cycloalkylene group includingone or more, e.g., 1-4, 1-3, 1, 2, 3, or 4, heteroatoms, e.g., N, O, andS. Examples of cycloalkylenes include, but are not limited to,cyclopropylene and cyclobutylene. A tetrahydrofuran may be considered asa heterocycloalkylene.

The term “arylene,” as used herein, refers to a multivalent (e.g.,divalent or trivalent) aryl group linking together multiple (e.g., twoor three) parts of a compound. For example, one carbon within thearylene group may be linked to one part of the compound, while anothercarbon within the arylene group may be linked to another part of thecompound. An arylene may have, e.g., five to fifteen carbons in the arylportion of the arylene (e.g., a C5-C6, C5-C7, C5-C8, C5-C9. C5-C10,C5-C11, C5-C12, C5-C13, C5-C14, or C5-C15 arylene). An arylene group canbe substituted by the groups typically suitable as substituents foralkyl, alkenyl and alkynyl groups as set forth herein. Heteroarylenerefers to an aromatic group including one or more, e.g., 1-4, 1-3, 1, 2,3, or 4, heteroatoms, e.g., N, O, and S. A heteroarylene group may have,e.g., two to fifteen carbons (e.g., a C2-C3, C2-C4, C2-C5, C2-C6, C2-C7,C2-C8, C2-C9. C2-C10, C2-C11, C2-C12, C2-C13, C2-C14, or C2-C15heteroarylene).

The term “optionally substituted,” as used herein, refers to having 0,1, or more substituents, such as 0-25, 0-20, 0-10 or 0-5 substituents.Substituents include, but are not limited to, alkyl, alkenyl, alkynyl,aryl, alkaryl, acyl, heteroaryl, heteroalkyl, heteroalkenyl,heteroalkynyl, heteroalkaryl, halogen, oxo, cyano, nitro, amino,alkamino, hydroxy, alkoxy, alkanoyl, carbonyl, carbamoyl, guanidinyl,ureido, amidinyl, any of the groups or moieties described above, andhetero versions of any of the groups or moieties described above.Substituents include, but are not limited to, F, Cl, methyl, phenyl,benzyl, OR, NR₂, SR, SOR, SO₂R, OCOR, NRCOR, NRCONR₂, NRCOOR, OCONR₂,RCO, COOR, alkyl-OOCR, SO₃R, CONR₂, SO₂NR₂, NRSO₂NR₂, CN, CF₃, OCF₃,SiR₃, and NO₂, wherein each R is, independently, H, alkyl, alkenyl,aryl, heteroalkyl, heteroalkenyl, or heteroaryl, and wherein two of theoptional substituents on the same or adjacent atoms can be joined toform a fused, optionally substituted aromatic or nonaromatic, saturatedor unsaturated ring which contains 3-8 members, or two of the optionalsubstituents on the same atom can be joined to form an optionallysubstituted aromatic or nonaromatic, saturated or unsaturated ring whichcontains 3-8 members.

An optionally substituted group or moiety refers to a group or moiety(e.g., any one of the groups or moieties described above) in which oneof the atoms (e.g., a hydrogen atom) is optionally replaced with anothersubstituent. For example, an optionally substituted alkyl may be anoptionally substituted methyl, in which a hydrogen atom of the methylgroup is replaced by, e.g., OH. As another example, a substituent on aheteroalkyl or its divalent counterpart, heteroalkylene, may replace ahydrogen on a carbon or a hydrogen on a heteroatom such as N. Forexample, the hydrogen atom in the group —R—NH—R— may be substituted withan alkamide substituent, e.g., —R—N[(CH₂C(O)N(CH₃)₂]—R. Generally, anoptional substituent is a non interfering substituent. A “noninterferingsubstituent” refers to a substituent that leaves the ability of theconjugates described herein (e.g., conjugates of any one of formulas(1)-(5), (D-I)-(D-X), (D′-I), (M-I)-(M-X), or (M′-I)) to either bind toviral neuraminidase or to inhibit the proliferation of influenza virus.Thus, in some embodiments, the substituent may alter the degree of suchactivity. However, as long as the conjugate retains the ability to bindto viral neuraminidase or to inhibitor viral proliferation, thesubstituent will be classified as “noninterfering.” For example, thenoninterfering substituent would leave the ability of the compound toprovide antiviral efficacy based on an IC50 value of 10 μM or less in aviral plaque reduction assay, such as in Example 2 based on an IC50value against influenza virus neuraminidase of less than 500 nM. Thus,the substituent may alter the degree of inhibition based on plaquereduction or influenza virus neuraminidase inhibition. However, as longas the compounds herein such as compounds of formulas (A-I), (A-II),(A-III), (A-IV), (A-V), (A-VI), (A-VII), (A-VIII), (A-IX), (A-X),(A-XI), and (A-XII) retain the ability to inhibit influenza virusneuraminidase activity, the substituent will be classified as“noninterfering.” A number of assays for determining viral plaquereduction or the ability of any compound to inhibit influenza virusneuraminidase are available in the art, and some are exemplified in theExamples below.

The term “hetero,” when used to describe a chemical group or moiety,refers to having at least one heteroatom that is not a carbon or ahydrogen, e.g., N, O, and S. Any one of the groups or moieties describedabove may be referred to as hetero if it contains at least oneheteroatom. For example, a heterocycloalkyl, heterocycloalkenyl, orheterocycloalkynyl group refers to a cycloalkyl, cycloalkenyl, orcycloalkynyl group that has one or more heteroatoms independentlyselected from, e.g., N, O, and S. An example of a heterocycloalkenylgroup is a maleimido. For example, a heteroaryl group refers to anaromatic group that has one or more heteroatoms independently selectedfrom, e.g., N, O, and S. One or more heteroatoms may also be included ina substituent that replaced a hydrogen atom in a group or moiety asdescribed herein. For example, in an optionally substituted heteroarylgroup, if one of the hydrogen atoms in the heteroaryl group is replacedwith a substituent (e.g., methyl), the substituent may also contain oneor more heteroatoms (e.g., methanol).

The term “acyl,” as used herein, refers to a group having the structure:

wherein R^(z) is an optionally substituted alkyl, alkenyl, alkynyl,cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, alkaryl, alkamino,heteroalkyl, heteroalkenyl, heteroalkynyl, heterocycloalkyl,heterocycloalkenyl, heterocycloalkynyl, heteroaryl, heteroalkaryl, orheteroalkamino.

The term “halo” or “halogen,” as used herein, refers to any halogenatom, e.g., F, Cl, Br, or I. Any one of the groups or moieties describedherein may be referred to as a “halo moiety” if it contains at least onehalogen atom, such as haloalkyl.

The term “hydroxyl,” as used herein, represents an —OH group.

The term “oxo,” as used herein, refers to a substituent having thestructure ═O, where there is a double bond between an atom and an oxygenatom.

The term “carbonyl,” as used herein, refers to a group having thestructure:

The term “thiocarbonyl,” as used herein, refers to a group having thestructure:

The term “phosphate,” as used herein, represents the group having thestructure:

The term “phosphoryl,” as used herein, represents the group having thestructure:

The term “sulfonyl,” as used herein, represents the group having thestructure:

The term “imino,” as used herein, represents the group having thestructure:

wherein R is an optional substituent.

The term “N-protecting group,” as used herein, represents those groupsintended to protect an amino group against undesirable reactions duringsynthetic procedures. Commonly used N-protecting groups are disclosed inGreene, “Protective Groups in Organic Synthesis,” 5th Edition (JohnWiley & Sons, New York, 2014), which is incorporated herein byreference. N-protecting groups include, e.g., acyl, aryloyl, andcarbamyl groups such as formyl, acetyl, propionyl, pivaloyl,t-butylacetyl, 2-chloroacetyl, 2-bromoacetyl, trifluoroacetyl,trichloroacetyl, phthalyl, o-nitrophenoxyacetyl, α-chlorobutyryl,benzoyl, carboxybenzyl (CBz), 4-chlorobenzoyl, 4-bromobenzoyl,4-nitrobenzoyl, and chiral auxiliaries such as protected or unprotectedD, L or D, L-amino acid residues such as alanine, leucine,phenylalanine; sulfonyl-containing groups such as benzenesulfonyl andp-toluenesulfonyl; carbamate forming groups such as benzyloxycarbonyl,p-chlorobenzyloxycarbonyl, p-methoxybenzyloxycarbonyl,p-nitrobenzyloxycarbonyl, 2-nitrobenzyloxycarbonyl,p-bromobenzyloxycarbonyl, 3,4-dimethoxybenzyloxycarbonyl,3,5-dimethoxybenzyl oxycarbonyl, 2,4-dimethoxybenzyloxycarbonyl,4-methoxybenzyloxycarbonyl, 2-nitro-4,5-dimethoxybenzyloxycarbonyl,3,4,5-trimethoxybenzyloxycarbonyl,1-(p-biphenylyl)-1-methylethoxycarbonyl,α,α-dimethyl-3,5-dimethoxybenzyloxycarbonyl, benzhydryloxy carbonyl,t-butyloxycarbonyl (BOC), diisopropylmethoxycarbonyl,isopropyloxycarbonyl, ethoxycarbonyl, methoxycarbonyl, allyloxycarbonyl,2,2,2,-trichloroethoxycarbonyl, phenoxycarbonyl, 4-nitrophenoxycarbonyl, fluorenyl-9-methoxycarbonyl (Fmoc), cyclopentyloxycarbonyl,adamantyloxycarbonyl, cyclohexyloxycarbonyl, and phenylthiocarbonyl;alkaryl groups such as benzyl, triphenylmethyl, and benzyloxymethyl; andsilyl groups such as trimethylsilyl.

The term “amino acid,” as used herein, means naturally occurring aminoacids and non-naturally occurring amino acids.

The term “naturally occurring amino acids,” as used herein, means aminoacids including Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, lie, Leu,Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, and Val.

The term “non-naturally occurring amino acid,” as used herein, means analpha amino acid that is not naturally produced or found in a mammal.Examples of non-naturally occurring amino acids include D-amino acids;an amino acid having an acetylaminomethyl group attached to a sulfuratom of a cysteine; a pegylated amino acid; the omega amino acids of theformula NH₂(CH₂)_(n)COOH where n is 2-6, neutral nonpolar amino acids,such as sarcosine, t-butyl alanine, t-butyl glycine, N-methylisoleucine, and norleucine; oxymethionine; phenylglycine; citrulline;methionine sulfoxide; cysteic acid; ornithine; diaminobutyric acid;3-aminoalanine; 3-hydroxy-D-proline; 2,4-diaminobutyric acid;2-aminopentanoic acid; 2-aminooctanoic acid, 2-carboxy piperazine;piperazine-2-carboxylic acid, 2-amino-4-phenylbutanoic acid;3-(2-naphthyl)alanine, and hydroxyproline. Other amino acids areα-aminobutyric acid, α-amino-α-methylbutyrate,aminocyclopropane-carboxylate, aminoisobutyric acid,aminonorbornyl-carboxylate, L-cyclohexylalanine, cyclopentylalanine,L-N-methylleucine, L-N-methylmethionine, L-N-methylnorvaline,L-N-methylphenylalanine, L-N-methylproline, L-N-methylserine,L-N-methyltryptophan, D-ornithine, L-N-methylethylglycine, L-norleucine,α-methyl-aminoisobutyrate, α-methylcyclohexylalanine, D-α-methylalanine,D-α-methylarginine, D-α-methylasparagine, D-α-methylaspartate,D-α-methylcysteine, D-α-methylglutamine, D-α-methylhistidine,D-α-methylisoleucine, D-α-methylleucine, D-α-methyllysine,D-α-methylmethionine, D-α-methylornithine, D-α-methylphenylalanine,D-α-methylproline, D-α-methylserine, D-N-methylserine,D-α-methylthreonine, D-α-methyltryptophan, D-α-methyltyrosine,D-α-methylvaline, D-N-methylalanine, D-N-methylarginine,D-N-methylasparagine, D-N-methylaspartate, D-N-methylcysteine,D-N-methylglutamine, D-N-methylglutamate, D-N-methylhistidine,D-N-methylisoleucine, D-N-methylleucine, D-N-methyllysine,N-methylcyclohexylalanine, D-N-methylornithine, N-methylglycine,N-methylaminoisobutyrate, N-(1-methylpropyl)glycine,N-(2-methylpropyl)glycine, D-N-methyltryptophan, D-N-methyltyrosine,D-N-methylvaline, γ-aminobutyric acid, L-t-butylglycine, L-ethylglycine,L-homophenylalanine, L-α-methylarginine, L-α-methylaspartate,L-α-methylcysteine, L-α-methylglutamine, L-α-methylhistidine,L-α-methylisoleucine, L-α-methylleucine, L-α-methylmethionine,L-α-methylnorvaline, L-α-methylphenylalanine, L-α-methylserine,L-α-methyltryptophan, L-α-methylvaline, N—(N-(2,2-diphenylethyl)carbamylmethylglycine,1-carboxy-1-(2,2-diphenyl-ethylamino)cyclopropane, 4-hydroxyproline,ornithine, 2-aminobenzoyl (anthraniloyl), D-cyclohexylalanine,4-phenyl-phenylalanine, L-citrulline, α-cyclohexylglycine,L-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid,L-thiazolidine-4-carboxylic acid, L-homotyrosine, L-2-furylalanine,L-histidine (3-methyl), N-(3-guanidinopropyl)glycine,O-methyl-L-tyrosine, O-glycan-serine, meta-tyrosine, nor-tyrosine,L-N,N′,N″-trimethyllysine, homolysine, norlysine, N-glycan asparagine,7-hydroxy-1,2,3,4-tetrahydro-4-fluorophenylalanine,4-methylphenylalanine, bis-(2-picolyl)amine, pentafluorophenylalanine,indoline-2-carboxylic acid, 2-aminobenzoic acid, 3-amino-2-naphthoicacid, asymmetric dimethylarginine, L-tetrahydroisoquinoline-1-carboxylicacid, D-tetrahydroisoquinoline-1-carboxylic acid, 1-amino-cyclohexaneacetic acid, D/L-allylglycine, 4-aminobenzoic acid, 1-amino-cyclobutanecarboxylic acid, 2 or 3 or 4-aminocyclohexane carboxylic acid,1-amino-1-cyclopentane carboxylic acid, 1-aminoindane-1-carboxylic acid,4-amino-pyrrolidine-2-carboxylic acid, 2-aminotetraline-2-carboxylicacid, azetidine-3-carboxylic acid, 4-benzyl-pyrolidine-2-carboxylicacid, tert-butylglycine, b-(benzothiazolyl-2-yl)-alanine, b-cyclopropylalanine, 5,5-dimethyl-1,3-thiazolidine-4-carboxylic acid,(2R,4S)4-hydroxypiperidine-2-carboxylic acid, (2S,4S) and(2S,4R)-4-(2-naphthylmethoxy)-pyrolidine-2-carboxylic acid, (2S,4S) and(2S,4R)4-phenoxy-pyrrolidine-2-carboxylic acid, (2R,5S) and(2S,5R)-5-phenyl-pyrrolidine-2-carboxylic acid,(2S,4S)-4-amino-1-benzoyl-pyrrolidine-2-carboxylic acid, t-butylalanine,(2S,5R)-5-phenyl-pyrrolidine-2-carboxylic acid,1-aminomethyl-cyclohexane-acetic acid, 3,5-bis-(2-amino)ethoxy-benzoicacid, 3,5-diamino-benzoic acid, 2-methylamino-benzoic acid,N-methylanthranylic acid, L-N-methylalanine, L-N-methylarginine,L-N-methylasparagine, L-N-methylaspartic acid, L-N-methylcysteine,L-N-methylglutamine, L-N-methylglutamic acid, L-N-methylhistidine,L-N-methylisoleucine, L-N-methyllysine, L-N-methylnorleucine,L-N-methylornithine, L-N-methylthreonine, L-N-methyltyrosine,L-N-methylvaline, L-N-methyl-t-butylglycine, L-norvaline,α-methyl-γ-aminobutyrate, 4,4′-biphenylalanine,α-methylcylcopentylalanine, α-methyl-α-napthylalanine,α-methylpenicillamine, N-(4-aminobutyl)glycine, N-(2-aminoethyl)glycine,N-(3-aminopropyl)glycine, N-amino-α-methylbutyrate, α-napthylalanine,N-benzylglycine, N-(2-carbamylethyl)glycine, N-(carbamylmethyl)glycine,N-(2-carboxyethyl)glycine, N-(carboxymethyl)glycine,N-cyclobutylglycine, N-cyclodecylglycine, N-cycloheptylglycine,N-cyclohexylglycine, N-cyclodecylglycine, N-cylcododecylglycine,N-cyclooctylglycine, N-cyclopropylglycine, N-cycloundecylglycine,N-(2,2-diphenylethyl)glycine, N-(3,3-diphenylpropyl)glycine,N-(3-guanidinopropyl)glycine, N-(1-hydroxyethyl)glycine,N-(hydroxyethyl))glycine, N-(imidazolylethyl))glycine,N-(3-indolylyethyl)glycine, N-methyl-γ-aminobutyrate,D-N-methylmethionine, N-methylcyclopentylalanine,D-N-methylphenylalanine, D-N-methylproline, D-N-methylthreonine,N-(1-methylethyl)glycine, N-methyl-napthylalanine,N-methylpenicillamine, N-(p-hydroxyphenyl)glycine,N-(thiomethyl)glycine, penicillamine, L-α-methylalanine,L-α-methylasparagine, L-α-methyl-t-butylglycine, L-methylethylglycine,L-α-methylglutamate, L-α-methylhomophenylalanine,N-(2-methylthioethyl)glycine, L-α-methyllysine, L-α-methylnorleucine,L-α-methylornithine, L-α-methylproline, L-α-methylthreonine,L-α-methyltyrosine, L-N-methylhomophenylalanine,N—(N-(3,3-diphenylpropyl) carbamylmethylglycine, L-pyroglutamic acid,D-pyroglutamic acid, O-methyl-L-serine, O-methyl-L-homoserine,5-hydroxylysine, α-carboxyglutamate, phenylglycine, L-pipecolic acid(homoproline), L-homoleucine, L-lysine (dimethyl), L-2-naphthylalanine,L-dimethyldopa or L-dimethoxy-phenylalanine, L-3-pyridylalanine,L-histidine (benzoyloxymethyl), N-cycloheptylglycine, L-diphenylalanine,O-methyl-L-homotyrosine, L-β-homolysine, O-glycan-threoine,Ortho-tyrosine, L-N,N′-dimethyllysine, L-homoarginine, neotryptophan,3-benzothienylalanine, isoquinoline-3-carboxylic acid, diaminopropionicacid, homocysteine, 3,4-dimethoxyphenylalanine, 4-chlorophenylalanine,L-1,2,3,4-tetrahydronorharman-3-carboxylic acid, adamantylalanine,symmetrical dimethylarginine, 3-carboxythiomorpholine,D-1,2,3,4-tetrahydronorharman-3-carboxylic acid, 3-aminobenzoic acid,3-amino-1-carboxymethyl-pyridin-2-one, 1-amino-1-cyclohexane carboxylicacid, 2-aminocyclopentane carboxylic acid, 1-amino-1-cyclopropanecarboxylic acid, 2-aminoindane-2-carboxylic acid,4-amino-tetrahydrothiopyran-4-carboxylic acid, azetidine-2-carboxylicacid, b-(benzothiazol-2-yl)-alanine, neopentylglycine, 2-carboxymethylpiperidine, b-cyclobutyl alanine, allylglycine, diaminopropionic acid,homo-cyclohexyl alanine, (2S,4R)-4-hydroxypiperidine-2-carboxylic acid,octahydroindole-2-carboxylic acid, (2S,4R) and (2S,4R)-4-(2-naphthyl),pyrrolidine-2-carboxylic acid, nipecotic acid, (2S,4R) and(2S,4S)-4-(4-phenylbenzyl) pyrrolidine-2-carboxylic acid,(3S)-1-pyrrolidine-3-carboxylic acid,(2S,4S)-4-tritylmercapto-pyrrolidine-2-carboxylic acid,(2S,4S)-4-mercaptoproline, t-butylglycine,N,N-bis(3-aminopropyl)glycine, 1-amino-cyclohexane-1-carboxylic acid,N-mercaptoethylglycine, and selenocysteine. In some embodiments, aminoacid residues may be charged or polar. Charged amino acids includealanine, lysine, aspartic acid, or glutamic acid, or non-naturallyoccurring analogs thereof. Polar amino acids include glutamine,asparagine, histidine, serine, threonine, tyrosine, methionine, ortryptophan, or non-naturally occurring analogs thereof. It isspecifically contemplated that in some embodiments, a terminal aminogroup in the amino acid may be an amido group or a carbamate group.

As used herein, the term “percent (%) identity” refers to the percentageof amino acid residues of a candidate sequence, e.g., an Fc-IgG, orfragment thereof, that are identical to the amino acid residues of areference sequence after aligning the sequences and introducing gaps, ifnecessary, to achieve the maximum percent identity (i.e., gaps can beintroduced in one or both of the candidate and reference sequences foroptimal alignment and non-homologous sequences can be disregarded forcomparison purposes). Alignment for purposes of determining percentidentity can be achieved in various ways that are within the skill inthe art, for instance, using publicly available computer software suchas BLAST, ALIGN, or Megalign (DNASTAR) software. Those skilled in theart can determine appropriate parameters for measuring alignment,including any algorithms needed to achieve maximal alignment over thefull length of the sequences being compared. In some embodiments, thepercent amino acid sequence identity of a given candidate sequence to,with, or against a given reference sequence (which can alternatively bephrased as a given candidate sequence that has or includes a certainpercent amino acid sequence identity to, with, or against a givenreference sequence) is calculated as follows:

100×(fraction of A/B)

where A is the number of amino acid residues scored as identical in thealignment of the candidate sequence and the reference sequence, andwhere B is the total number of amino acid residues in the referencesequence. In some embodiments where the length of the candidate sequencedoes not equal to the length of the reference sequence, the percentamino acid sequence identity of the candidate sequence to the referencesequence would not equal to the percent amino acid sequence identity ofthe reference sequence to the candidate sequence.

Two polynucleotide or polypeptide sequences are said to be “identical”if the sequence of nucleotides or amino acids in the two sequences isthe same when aligned for maximum correspondence as described above.Comparisons between two sequences are typically performed by comparingthe sequences over a comparison window to identify and compare localregions of sequence similarity. A “comparison window” as used herein,refers to a segment of at least about 15 contiguous positions, about 20contiguous positions, about 25 contiguous positions, or more (e.g.,about 30 to about 75 contiguous positions, or about 40 to about 50contiguous positions), in which a sequence may be compared to areference sequence of the same number of contiguous positions after thetwo sequences are optimally aligned.

The term “treating” or “to treat,” as used herein, refers to atherapeutic treatment of a viral infection (e.g., a viral infection suchas and influenza infection) in a subject. In some embodiments, atherapeutic treatment may slow the progression of the viral infection,improve the subject's outcome, and/or eliminate the infection. In someembodiments, a therapeutic treatment of a viral infection in a subjectmay alleby way ofte or ameliorate of one or more symptoms or conditionsassociated with the viral infection, diminish the extent of the viral,stabilize (i.e., not worsening) the state of the viral infection,prevent the spread of the viral infection, and/or delay or slow theprogress of the viral infection, as compare the state and/or thecondition of the viral infection in the absence of the therapeutictreatment.

The term “average value of T,” as used herein, refers to the mean numberof monomers of neuraminidase inhibitor or dimers of neuramindaseinhibitors conjugated to an Fc domain or an albumin protein within apopulation of conjugates. In some embodiments, within a population ofconjugates, the average number of monomers of neuraminidase inhibitor ordimers of neuramindase inhibitors conjugated to an Fc domain monomer maybe from 1 to 20 (e.g., the average value of T is 1 to 2, 1 to 3, 1 to 4,1 to 5, 5 to 10, 10 to 15, or 15 to 20). In some embodiments, theaverage value of T is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, or 20.

The term “subject,” as used herein, can be a human, non-human primate,or other mammal, such as but not limited to dog, cat, horse, cow, pig,turkey, goat, fish, monkey, chicken, rat, mouse, and sheep.

The term “therapeutically effective amount,” as used herein, refers toan amount, e.g., pharmaceutical dose, effective in inducing a desiredeffect in a subject or in treating a subject having a condition ordisorder described herein (e.g., a viral infection, such as an influezainfection). It is also to be understood herein that a “therapeuticallyeffective amount” may be interpreted as an amount giving a desiredtherapeutic and/or preventative effect, taken in one or more doses or inany dosage or route, and/or taken alone or in combination with othertherapeutic agents (e.g., an antiviral agent described herein). Forexample, in the context of administering a conjugate described herein(e.g., a conjugate of any one of formulas (1)-(5), (D-I)-(D-X), (D′-I),(M-I)-(M-X), or (M′-I)) that is used for the treatment of a viralinfection, an effective amount of a conjugate is, for example, an amountsufficient to prevent, slow down, or reverse the progression of theviral infection as compared to the response obtained withoutadministration of the conjugate.

As used herein, the term “pharmaceutical composition” refers to amedicinal or pharmaceutical formulation that contains at least oneactive ingredient (e.g., a conjugate of any one of formulas (1)-(5),(D-I)-(D-X), (D′-I), (M-I)-(M-X), or (M′-I)) as well as one or moreexcipients and diluents to enable the active ingredient suitable for themethod of administration. The pharmaceutical composition of the presentdisclosure includes pharmaceutically acceptable components that arecompatible with a conjugate described herein (e.g., a conjugate of anyone of formulas (1)-(5), (D-I)-(D-X), (D′-I), (M-I)-(M-X), or (M′-I)).

As used herein, the term “pharmaceutically acceptable carrier” refers toan excipient or diluent in a pharmaceutical composition. For example, apharmaceutically acceptable carrier may be a vehicle capable ofsuspending or dissolving the active conjugate (e.g., a conjugate of anyone of formulas (1)-(5), (D-I)-(D-X), or (M-I)-(M-VI)). Thepharmaceutically acceptable carrier must be compatible with the otheringredients of the formulation and not deleterious to the recipient. Inthe present disclosure, the pharmaceutically acceptable carrier mustprovide adequate pharmaceutical stability to a conjugate describedherein. The nature of the carrier differs with the mode ofadministration. For example, for oral administration, a solid carrier ispreferred; for intravenous administration, an aqueous solution carrier(e.g., WFI, and/or a buffered solution) is generally used.

The term “pharmaceutically acceptable salt,” as used herein, representssalts of the conjugates described herein (e.g., conjugates of any one offormulas (1)-(5), (D-I)-(D-X), (D′-I), (M-I)-(M-X), or (M′-I)) that are,within the scope of sound medical judgment, suitable for use in methodsdescribed herein without undue toxicity, irritation, and/or allergicresponse. Pharmaceutically acceptable salts are well known in the art.For example, pharmaceutically acceptable salts are described in:Pharmaceutical Salts: Properties, Selection, and Use (Eds. P. H. Stahland C. G. Wermuth), Wiley-VCH, 2008. The salts can be prepared in situduring the final isolation and purification of the conjugates describedherein or separately by reacting the free base group with a suitableorganic acid.

The term “drug-to-antibody ratio” or “DAR” refers to the average numberof small molecule drug moieties (e.g., the average number of smallmolecule drug monomers or dimers) conjugated to an antibody, Fc domain,or albumin protein described herein. In some embodiments describedherein, the DAR is represented by “T” (e.g., in formulas (1)-(5),(D-I)-(D-X), (D′-I), (M-I)-(M-X), or (M′-I)). As used herein, eachmonomer moiety (e.g., each zanamivir or peramivir monomer) or each dimermoiety (e.g., each zanamivir dimer or peramivir dimer) conjugated to theFc domain, antibody, or albumin protein corresponds to a DAR value of1.0 (e.g., a “T” value of 1.0). For example, an Fc domain conjugated to4 zanamivir monomers would have a DAR of 4.0 (e.g., a “T” of 4.0). An Fcdomain conjugated to 4 zanamivir dimers (e.g., 8 total zanamivirmolecules) would also have a DAR of 4.0 (e.g., a “T” of 4.0). DAR mayalso be computed as the average DAR for a population of molecules, suchas a population of Fc domains, antibodies, or albumin proteins. DARvalues may affect the efficacy, potency, pharmacokinetics, or toxicityof the drug.

The term “about,” as used herein, indicates a deby way oftion of ±5%.For example, about 10% refers to from 9.5% to 10.5%.

Any values provided in a range of values include both the upper andlower bounds, and any values contained within the upper and lowerbounds.

The term “(1)-(5), (D-I)-(D-X), (D′-I), (M-I)-(M-X), or (M′-I)”, as usedherein, represents the formulas of any one of (1), (2), (3), (4), (5),(D-I), (D-II), (D-II-1), (D-II-2), (D-II-3), (D-II-4), (D-II-5),(D-II-6), (D-II-7), (D-II-8), (D-II-9), (D-II-10), (D-III), (D-III-1),(D-III-2), (D-III-3), (D-III-4), (D-III-5), (D-III-6), (D-III-7),(D-III-8), (D-III-9), (D-IV), (D-IV-1), (D-IV-2), (D-V), (D-V-1),(D-V-2), (D-V-3), (D-V-4), (D-V-5), (D-V-6), (D-V-7), (D-V-8), (D-V-9),(D-V-10), (D-VI), (D-VI-1), (D-VI-2), (D-VI-3), (D-VI-4), (D-VI-5),(D-VI-6), (D-VI-7), (D-VI-8), (D-VI-9), (D-VII), (D-VIII), (D-VIII-1),(D-VIII-2), (D-VIII-3), (D-VIII-4), (D-VIII-5), (D-VIII-6), (D-VIII-7),(D-VIII-8), (D-VIII-9), (D-VIII-10), (D-VIII-11), (D-IX), (D-IX-1),(D-IX-2), (D-IX-3), (D-IX-4), (D-IX-5), (D-IX-6), (D-X), (D-X-1),(D-X-2), (D-X-3), (D′-I), (M-I), (M-II), (M-II-1), (M-II-2), (M-II-3),(M-II-4), (M-II-5), (M-II-6), (M-II-7), (M-II-8), (M-II-9), (M-II-10),(M-III), (M-III-1), (M-III-2), (M-III-3), (M-III-4), (M-III-5),(M-III-6), (M-III-7), (M-III-8), (M-III-9), (M-IV), (M-IV-1), (M-IV-2),(M-V), (M-V-1), (M-V-2), (M-V-3), (M-V-4), (M-V-5), (M-V-6), (M-V-7),(M-V-8), (M-V-9), (M-V-10), (M-VI), (M-VI-1), (M-VI-2), (M-VI-3),(M-VI-4), (M-VI-5), (M-VI-6), (M-VI-7), (M-VI-8), (M-VI-9), (M-VII),(M-VIII), (M-VIII-1), (M-VIII-2), (M-VIII-3), (M-VIII-4), (M-VIII-5),(M-VIII-6), (M-VIII-7), (M-VIII-8), (M-VII I-9), (M-VIII-10),(M-VIII-11), (M-IX), (M-IX-1), (M-IX-2), (M-IX-3), (M-IX-4), (M-IX-5),(M-IX-6), (M-X), (M-X-1), (M-X-2), (M-X-3), or (M′-I)).

Other features and advantages of the conjugates described herein will beapparent from the following Detailed Description and the claims.

DESCRIPTION OF THE DRAWINGS

FIG. 1 is an image depicting exemplary methods of conjugating aneuraminidase inhibitor monomer or dimer, e.g., by way of a linker, toan Fc domain monomer, an Fc domain, an Fc-binding peptide, an albuminprotein, or an albumin protein-binding peptide.

FIG. 2 shows non-reducing and reducing SDS-PAGE and a schematicillustration of an Fc domain formed from Fc domain monomers having thesequence of SEQ ID NO: 1.

FIG. 3 shows non-reducing and reducing SDS-PAGE and a schematicillustration of an Fc domain formed from Fc domain monomers having thesequence of SEQ ID NO: 3.

FIG. 4 shows non-reducing and reducing SDS-PAGE and a schematicillustration of an Fc domain formed from Fc domain monomers having thesequence of SEQ ID NO: 5.

FIG. 5 shows non-reducing and reducing SDS-PAGE and a schematicillustration of an Fc domain formed from Fc domain monomers having thesequence of SEQ ID NO: 7.

FIG. 6 shows non-reducing and reducing SDS-PAGE and a schematicillustration of an Fc domain formed from Fc domain monomers having thesequence of SEQ ID NO: 9.

FIG. 7 shows non-reducing and reducing SDS-PAGE and a schematicillustration of an Fc domain formed from Fc domain monomers having thesequence of SEQ ID NO: 12.

FIG. 8 shows non-reducing and reducing SDS-PAGE and a schematicillustration of an Fc domain formed from Fc domain monomers having thesequence of SEQ ID NO: 14.

FIG. 9 shows a non-reducing SDS-PAGE of Conjugate 1.

FIG. 10 shows a non-reducing SDS-PAGE of Conjugate 2.

FIG. 11 shows a non-reducing SDS-PAGE of Conjugate 3.

FIG. 12 shows a non-reducing SDS-PAGE of Conjugate 4.

FIG. 13 shows a non-reducing SDS-PAGE of Conjugate 5.

FIG. 14 shows a non-reducing SDS-PAGE of Conjugate 6.

FIG. 15 is a graph showing the IC50 values from an H1N1 neuraminidaseinhibition assay for Int-2 and Conjugate 1.

FIG. 16 is a graph showing the IC50 values from an H1N1 neuraminidaseinhibition assay for Conjugates 1-6.

FIG. 17 is a graph showing the IC50 values from an H3N2 neuraminidaseinhibition assay for Conjugates 1-6.

FIGS. 18A-18C are a series of graphs showing the cell by way ofbility ofA549 cells treated with Conjugate 3 (FIG. 18A), Conjugate 4 (FIG. 18B),or Conjugate 6 (FIG. 18C).

FIGS. 19A-19E are a series of graphs showing the ability of Conjugate 3to inhibit the growth of pathogenic influenza viral strains A/WSN/33H1N1 (FIG. 19A), A/Wyoming/3/03 H3N2 (FIG. 19B), A/California/04/09 H1N1pdm (FIG. 19C), A/Vietnam/1203/04 H5N1 HALo (FIG. 19D), or B/Lee/40Victoria (FIG. 19E) in human epithelial cells.

FIGS. 20A-20E are a series of graphs showing the ability of Conjugate 4to inhibit the growth of pathogenic influenza viral strains A/WSN/33H1N1 (FIG. 20A), A/Wyoming/3/03 H3N2 (FIG. 20B), A/California/04/09 H1N1pdm (FIG. 20C), A/Vietnam/1203/04 H5N1 HALo (FIG. 20D), or B/Lee/40Victoria (FIG. 20E) in human epithelial cells.

FIGS. 21A-21E are a series of graphs showing the ability of Conjugate 6to inhibit the growth of pathogenic influenza viral strains A/WSN/33H1N1 (FIG. 21A), A/Wyoming/3/03 H3N2 (FIG. 21B), A/California/04/09 H1N1pdm (FIG. 21C), A/Vietnam/1203/04 H5N1 HALo (FIG. 21D), or B/Lee/40Victoria (FIG. 21E) in human epithelial cells.

FIGS. 22A-22E are a series of graphs showing the ability of Conjugate 6to inhibit the growth of pathogenic influenza viral strains A/WSN/33H1N1 (FIG. 22A), A/Wyoming/3/03 H3N2 (FIG. 22B), A/California/04/09 H1N1pdm (FIG. 22C), B/Lee/40 Victoria (FIG. 22D), or A/Vietnam/1203/04 (FIG.22E) in human epithelial cells, compared to Oseltamivir.

FIG. 23 is a graph showing the relative mouse serum concentration ofConjugate 6 compared to Fc (hIgG1) alone.

FIG. 24 is a graph showing the effect of Conjugate 6 on mouse weight ina lethal mouse influenza model. The study was performed as described inExample 29.

FIG. 25 is a graph showing the effect of Conjugate 6 on survival in alethal mouse influenza model. The study was performed as described inExample 29.

FIG. 26 is a graph showing the effect of Conjugate 6 on mouse weight ina lethal mouse influenza model. The study was performed as described inExample 30.

FIG. 27 is a graph showing the effect of Conjugate 6 on survival in alethal mouse influenza model. The study was performed as described inExample 30.

FIG. 28 is an image depicting a method of conjugating a neuraminidaseinhibitor monomer or dimer, e.g., by way of a linker, to an Fc domainmonomer, an Fc domain, an Fc-binding peptide, an albumin protein, or analbumin protein-binding peptide by oxime conjugation to an amino acidresidue, e.g., a nitrogen atom of a surface exposed lysine.

FIG. 29 is an image depicting a method of conjugating a neuraminidaseinhibitor monomer or dimer, e.g., by way of a linker, to an Fc domainmonomer, an Fc domain, an Fc-binding peptide, an albumin protein, or analbumin protein-binding peptide by thioether conjugation to an aminoacid residue, e.g., a nitrogen atom of a surface exposed lysine.

FIG. 30 is an image depicting a method of conjugating a neuraminidaseinhibitor monomer or dimer, e.g., by way of a linker, to an Fc domainmonomer, an Fc domain, an Fc-binding peptide, an albumin protein, or analbumin protein-binding peptide by rebridged cysteine conjugation, e.g.,rebridged cysteine conjugation to a pair of sulfur atoms of two hingecysteines in an Fc domain monomer or Fc domain.

FIGS. 31A-31F are a series of graphs showing showing the survival ofmice treated with Conjugate 6 in a lethal mouse influenza model. Micewere treated with either Oseltamivir (Tamiflu™) control, 20 mg/kg, 2×daily, starting 8 hours post-infection (FIG. 31A); Conjugate 6, 50mg/kg, 1 dose 28 days prior to infection (FIG. 31B); Conjugate 6, 10mg/kg, 1 dose 28 days prior to infection (FIG. 31C); Conjugate 6, 5mg/kg, 1 dose 28 days prior to infection (FIG. 31D); Conjugate 6, 2.5mg/kg, 1 dose 28 days prior to infection (FIG. 31E); or Conjugate 6,1.25 mg/kg, 1 dose 28 days prior to infection (FIG. 31F). This study wasperformed as described in Example 33.

FIGS. 32A-32F is a series of graphs showing showing that Conjugate 6extends treatment window as compared to Oseltamivir (Tamiflu™) asdetermined by survival in a lethal mouse influenza model. Mice weretreated with either vehicle (PBS), Fc only 10 mpk, or Conjugate 6, 4hours prior to inflection (FIG. 32A); Conjugate 6 or Oseltamivir(Tamiflu™), 8 hours post-infection (FIG. 32B); Conjugate 6 orOseltamivir (Tamiflu™), 24 hours post-infection (FIG. 32C); Conjugate 6or Oseltamivir (Tamiflu™), 48 hours post-infection (FIG. 32D); Conjugate6 or Oseltamivir (Tamiflu™), 72 hours post-infection (FIG. 32E); orConjugate 6 or Oseltamivir (Tamiflu™), 96 hours post-infection (FIG.32F). The study was performed as described in Example 34.

FIG. 33 is a graph showing no significant effect of body weight gainwere observed following administration of Conjugate 6 in a 14 day ratdose-range finder toxicity study. The study was performed as describedin Example 35.

FIGS. 34A-34D is a series of graphs showing showing that Conjugate 6extends treatment window as compared to Oseltamivir (Tamiflu™) asdetermined by survival in a lethal mouse influenza model. Mice weretreated with either Oseltamivir (Tamiflu™) control, 20 mg/kg, 2× daily,starting 8 hours post-infection (FIG. 34A); Conjugate 6, 10 mg/kg, 1dose 4 hours prior to infection (FIG. 34B); Conjugate 6, 2 mg/kg, 1 dose4 hours prior to infection (FIG. 34C); or Conjugate 6, 0.4 mg/kg, 1 dose4 days prior to infection (FIG. 34D). The study was performed asdescribed in Example 37.

FIG. 35 is a graph showing the effect of Conjugate 6 on mouse weight ina lethal mouse influenza model. The study was performed as described inExample 37.

FIG. 36 is a graph showing a 7-day rat pharmacokinetic study followingIV administration of Conjugate 6. The study was performed as describedin Example 38.

FIG. 37 is a graph showing a 14-day rat pharmacokinetic study followingIV administration of Conjugate 6. The study was performed as describedin Example 39.

FIG. 38 is a graph showing a 28-day rat pharmacokinetic study comparingIV and SC administration of Conjugate 6. The study was performed asdescribed in Example 40.

FIG. 39 is a graph showing a 28-day non-human primate pharmacokineticstudy following IV administration of Conjugate 6. The study wasperformed as described in Example 41.

FIG. 40 is a graph showing a mouse lung distribution pharmacokineticstudy following IV administration of Conjugate 6. The study wasperformed as described in Example 42.

FIG. 41 is a graph showing a 5-day mouse pharmacokinetic study comparingIV, SC and IM administration of Conjugate 6. The study was performed asdescribed in Example 43.

FIG. 42 shows a non-reducing SDS-PAGE of Conjugate 8.

FIG. 43 shows the structure of Conjugate 6.

FIG. 44 is a graph showing a 24 hour in vitro mouse plasma stabilitystudy comparing Conjugate 6 incubated at 37° C. for 24 hr compared to acontrol and neat compound.

FIG. 45 is a graph showing a 24 hour in vitro human plasma stabilitystudy comparing Conjugate 6 incubated at 37° C. for 24 hr compared to acontrol and neat compound.

FIG. 46 is a graph showing a 24 hour mouse liver microsomal stabilitystudy comparing Conjugate 6 incubated at 37° C. for 24 hr in mouse livermicrosomal cells and heat killed mouse liver microsomal cells as acontrol.

FIG. 47 is a graph showing a 24 hour human liver microsomal stabilitystudy comparing Conjugate 6 incubated at 37° C. for 24 hr in human livermicrosomal cells and heat killed mouse liver microsomal cells as acontrol.

FIG. 48 is a graph showing the % body weight change in mice over 15 dayspost viral challenge. The study was performed as described in Example66.

FIG. 49 is a graph showing the % survival in mice over 15 days postviral challenge. The study was performed as described in Example 66.

FIG. 50 is a graph showing the binding of Conjugate 6 and Conjugate 12compared to hIgG1 Fc (WT) and hIgG1 Fc (N297A) to Fcγ receptor IIIA. Thestudy was performed as described in Example 67.

FIG. 51 is a graph showing the binding of Conjugate 6 and Conjugate toFcγ receptor IIIA. The study was performed as described in Example 67.

FIG. 52 is a graph showing a 7-day non-human primate toxicokinetic studyfollowing IV administration of Conjugate 6. The study was performed asdescribed in Example 68.

FIG. 53 is a graph showing a 28-day non-human primate pharmacokineticstudy comparing IV and SC administration of Conjugate 6. The study wasperformed as described in Example 68.

FIG. 54 shows a non-reducing SDS-PAGE of Conjugate 12.

FIG. 55 shows a non-reducing SDS-PAGE of Conjugate 13 having differentdrug-to-antibody (DAR) ratio values (Conjugate 13a, Conjugate 13b,Conjugate 13c, Conjugate 13d, Conjugate 13e, Conjugate 13f, andConjugate 13g).

FIG. 56 is a graph showing the % survival in immune compromised miceover 35 days post viral challenge. The 0.3 mg/kg conjugate 6 treatmentgroup remained at 100% survival but is slightly offset in the graph forclarity. The study was performed as described in Example 95

FIG. 57 is a graph showing the % body weight change in immunecompromised mice over 35 days post viral challenge. The study wasperformed as described in Example 95.

FIG. 58 is a graph showing administration of conjugate 6 results indose-dependent viral clearance in the lungs of a mouse model infectedwith Influenza A (H1N1) and that this viral clearance is greater thanPBS control, Fc-only control, or Oseltamivir control. This study wasperformed as described in Example 96.

FIG. 59 is a graph showing that administration of conjugate 6 results ina dose-dependent reduction in inflammatory cytokines in the lungs in amouse model infected with Influenza A (H1N1). This study was performedas described in Example 97.

FIG. 60 is a graph showing the pharmacokinetics of conjugate 6 in BALB/cSCID (immunocompromised) mice and CD-1 mice (immunocompetent). Thisstudy was performed as described in Example 98.

FIG. 61 is an image depicting conjugate 33.

FIGS. 62A-62B are graphs showing the body weight change (%) in BALB/cmice challenged intranasally with 3× the LD₉₅ of mouse adapted influenzaA.PR/8/1934 (H1N1). This study was performed as described in Example133.

FIG. 63A is a graph showing the viral burden on day 4 post infection.This study was performed as described in Example 133.

FIG. 63B is a graph showing the log reduction in viral burden on day 4post infection. This study was performed as described in Example 133.

FIG. 64A is a graph showing administration of conjugate 33 results indose-dependent reduction of viral burden in a mouse model infected withInfluenza A (H1N1) compared with PBS control or Fc-only control. Thisstudy was performed as described in Example 133.

FIG. 64B is a graph showing the log reduction in viral burden on day 4post infection. This study was performed as described in Example 133.

FIGS. 65A-65E are a series bar graphs showing administration ofconjugate 33 results in dose-dependent fold-reduction in cytokine levelsfor TNF-α (FIG. 65A), IL-6 (FIG. 65B), INF-γ (FIG. 65C), MCP-1 (FIG.65D), and MIP-1α (FIG. 65E). This study was performed as described inExample 133.

FIG. 66 is a chromatograph showing the stability of Int-80 compared toInt-4 on day 7 of incubating at 37° C. and 60° C. This study wasperformed as describe in Example 142.

FIG. 67 is a graph showing serial passage of A/CA/09 pdm in the presenceof Coonjugate 6, oseltamivir, baloxavir or PBS control in A549 cells toevaluate the potential for development of drug resistant mutant viralstrains under selective pressure with viral inhibitors. This study wasperformed as described in Example 147.

FIG. 68 is a graph showing serial passage of A/WSN/1933 in the presenceof Conjugate 6, Conjugate 33, oseltamivir, baloxavir or PBS control inMDCK cells to evaluate the potential for development of drug resistantmutant viral strains under selective pressure with viral inhibitors.This study was performed as described in Example 147.

DETAILED DESCRIPTION

The disclosure features conjugates, compositions, and methods for thetreatment of viral infections (e.g., influenza viral infections). Theconjugates disclosed herein include monomers or dimers of viralneuraminidase inhibitors (e.g., zanamivir, peramivir, or analogsthereof) conjugated to Fc monomers, Fc domains, Fc-binding peptides,albumin proteins, or albumin protein-binding peptides. The neuraminidaseinhibitor (e.g., zanamivir, peramivir, or analogs thereof) in theconjugates targets neuraminidase on the surface of the viral particle.The Fc monomers or Fc domains in the conjugates bind to FcγRs (e.g.,FcRn, FcγRI, FcγRIIa, FcγRIIc, FcγRIIIa, and FcγRIIIb) on immune cells,e.g., neutrophils, to activate phagocytosis and effector functions, suchas antibody-dependent cell-mediated cytotoxicity (ADCC), thus leading tothe engulfment and destruction of viral particles by immune cells andfurther enhancing the antiviral activity of the conjugates. The albuminor albumin-binding peptide may extend the half-life of the conjugate,for example, by binding of albumin to the recycling neonatal Fcreceptor. Such compositions are useful in methods for the inhibition ofviral growth and in methods for the treatment of viral infections, suchas those caused by an influenza virus A, influenza virus B and influenzavirus C.

I. Viral Infections

The compounds and pharmaceutical compositions described herein (e.g., aconjugate of any one of formulas (1)-(5), (D-I)-(D-X), (D′-I),(M-I)-(M-X), or (M′-I)) can be used to treat a viral infection (e.g., aninfluenza viral infection, such as influenza A, B, C, or parainfluenza).

Viral infection refers to the pathogenic growth of a virus (e.g., theinfluenza virus) in a host organism (e.g., a human subject). A viralinfection can be any situation in which the presence of a viralpopulation(s) is damaging to a host body. Thus, a subject is sufferingfrom a viral infection when an excessive amount of a viral population ispresent in or on the subject's body, or when the presence of a viralpopulation(s) is damaging the cells or other tissue of the subject.

Influenza, commonly known as “the flu”, is an infectious disease causedby an influenza virus. Symptoms can be mild to severe. The most commonsymptoms include: a high fever, runny nose, sore throat, muscle pains,headache, coughing, and feeling tired. These symptoms typically begintwo days after exposure to the virus and most last less than a week. Thecough, however, may last for more than two weeks. In children, there maybe nausea and vomiting, but these are less common in adults.Complications of influenza may include viral pneumonia, secondarybacterial pneumonia, sinus infections, and worsening of previous healthproblems such as asthma or heart failure. Sever complications may occurin subjects having weakened immune systems, such as the young, the old,those with illnesses that weaken the immune system, and those undergoingtherapy treatment resulting in a weakening of the immune system.

Three types of influenza viruses affect human subjects, namely Type A,Type B, and Type C. Usually, the virus is spread through the air fromcoughs or sneezes. This is believed to occur mostly over relativelyshort distances. It can also be spread by touching surfaces contaminatedby the virus and then touching the mouth or eyes. A person may beinfectious to others both before and during the time they are showingsymptoms. The infection may be confirmed by testing the throat, sputum,or nose for the virus. A number of rapid tests are available; however,people may still have the infection if the results are negative. A typeof polymerase chain reaction that detects the virus's RNA may be used todiagnose influenza infection.

II. Conjugates of the Disclosure

Provided herein are synthetic conjugates useful in the treatment ofviral infections (e.g., influenza infections). The conjugates disclosedherein include an Fc domain or an albumin protein conjugated to one ormore monomers neuraminidase inhibitors or one or more dimers of twoneuraminidase inhibitors (e.g., neuraminidase inhibitors selected fromzanamivir, sulfozanamivir, peramivir, A-315675, or analogs thereof). Thedimers of two neuraminidase inhibitors include a neuraminidase inhibitor(e.g., a first neuraminidase inhibitor of formula (A-I), (A-II),(A-III), (A-IV), (A-V), (A-VI), (A-VII), (A-VIII), (A-IX), (A-X),(A-XI), or (A-XII)) and a second neuraminidase inhibitor (e.g., a secondneuraminidase inhibitor of formula (A-I), (A-II), (A-III), (A-IV),(A-V), (A-VI), (A-VII), (A-VIII), (A-IX), (A-X), (A-XI), or (A-XII)).The first and second neuraminidase inhibitors are linked to each otherbyway of a linker.

Without being bound by theory, in some aspects, conjugates describedherein bind to the surface of a viral particle (e.g., bind to viralneuraminidase enzyme on the surface on an influenza viral particle)through the interactions between the neuraminidase inhibitor moieties inthe conjugates and proteins on the surface of the viral particle. Theneuraminidase inhibitor disrupts neuraminidase, an envelope glycoproteinthat cleaves sialic acids, i.e., terminal neuraminic acid residues, fromglycan structures on the surface of infected host cells, releasingprogeny viruses and allowing the spread of the virus from the host cellto uninfected surrounding cells.

Conjugates of the invention include neuraminidase inhibitor monomers anddimers conjugated to an Fc domain, Fc monomer, or Fc-binding peptide.The Fc domain in the conjugates described herein binds to the FcγRs(e.g., FcRn, FcγRI, FcγRIIa, FcγRIIc, FcγRIIIa, and FcγRIIIb) on immunecells. The binding of the Fc domain in the conjugates described hereinto the FcγRs on immune cells activates phagocytosis and effectorfunctions, such as antibody-dependent cell-mediated cytotoxicity (ADCC),thus leading to the engulfment and destruction of viral particles byimmune cells and further enhancing the antiviral activity of theconjugates.

Conjugates of the invention include neuraminidase inhibitor monomers anddimers conjugated to an albumin protein or an albumin protein-bindingpeptide. The albumin protein or albumin protein-binding peptide mayextend the half-life of the conjugate, for example, by binding ofalbumin to the recycling neonatal Fc receptor.

Conjugates provided herein are described by any one of formulas (1)-(5),(D-I)-(D-X), (D′-I), (M-I)-(M-X), or (M′-I). In some embodiments, theconjugates described herein include one or more monomers ofneuraminidase inhibitors conjugated to an Fc domain or an albuminprotein. In some embodiments, the conjugates described herein includeone or more dimers of neuraminidase inhibitors conjugated to an Fcdomain or an albumin protein. In some embodiments, when n is 2, E (an Fcdomain monomer) dimerizes to form an Fc domain.

Conjugates described herein may be synthesized using available chemicalsynthesis techniques in the art. In cases where a functional group isnot available for conjugation, a molecule may be derivatized usingconventional chemical synthesis techniques that are well known in theart. In some embodiments, the conjugates described herein contain one ormore chiral centers. The conjugates include each of the isolatedstereoisomeric forms as well as mixtures of stereoisomers in varyingdegrees of chiral purity, including racemic mixtures. It alsoencompasses the various diastereomers, enantiomers, and tautomers thatcan be formed.

Neuraminidase Inhibitors

A component of the conjugates described herein is an influenza virusneuraminidase inhibitor moiety. An influenza virus neuraminidaseinhibitor disrupts neuraminidase, an envelope glycoprotein that cleavessialic acids, i.e., terminal neuraminic acid residues, from glycanstructures on the surface of infected host cells, releasing progenyviruses and allowing the spread of the virus from the host cell touninfected surrounding cells. Examples of an influenza virusneuraminidase inhibitor include zanamivir (Relenza), sulfozanamivir,A-315675 and peramivir. In addition, derivatives of zanamivir,sulfozanamivir, A-315675 and peramivir, such as those found in theliterature, have neuraminidase inhibitor activity and are useful asneuraminidase inhibitor moieties of the compounds herein (see, forexample, Hadhazi et al. A sulfozanamivir analogue has potentanti-influenza virus activity. ChemMedChem Comm. 13:785-789 (2018) andIn vitro characterization of A-315675, a highly potent inhibitor of Aand B strain of influenze virus neuraminidases and influenza virusreplication. Antimicrobial Agents and Chemotherapy 46(4):1014-1021(2002)).

Conjugates described herein are separated into two types: (1) one ormore dimers of neuraminidase inhibitors conjugated to an Fc domain or analbumin protein and (2) one or more monomers of neuraminidase inhibitorsconjugated to an Fc domain or an albumin protein. The dimers ofneuraminidase inhibitors are linked to each other by way of a linker,such as the linkers described herein.

Viral neuraminidase inhibitors of the invention include zanamivir,sulfozanamivir, A-315675, peramavir, and analogs thereof, such as theviral neuraminidase inhibitors of formulas (A-I)-(A-XII):

wherein R₁ is selected from —OH, —NH₂, —NHC(═NH)NH₂, and —NHC(═NH)NHR₆;R₂ and R₃ are each independently selected from —H, —OH, —F, —Cl, and—Br; R₄ is selected from —CO₂H, —P(═O)(OH)₂, —SO₃H; R₅ is selected from—COCH₃, —COCF₃, —SO₂CH₃; X is selected from —O— and —S—; Y is selectedfrom

R₆ is selected from

R₇ is selected from H, C1-C20 alkyl, C3-C20 cycloalkyl, C3-C20heterocycloalkyl; C5-C15 aryl, and C2-C15 heteroaryl; and R₈ is selectedfrom C3-C20 heterocycloalkyl, C5-C15 aryl, and C2-C15 heteroaryl. Mostpreferably the viral neuraminidase inhibitor of formula (A-I), (A-II),(A-III), (A-IV), (A-V), (A-VI), (A-VII), (A-VIII), (A-IX), (A-X),(A-XI), or (A-XII) is covalently attached to the conjugate through Y.

Preferably the viral neuraminidase inhibitor is selected from zanamivir,sulfozanamivir, peramivir, or A-315675:

Conjugates of Dimers of Neuraminidase Inhibitors Linked to an Fc Domainor an Albumin Protein

The conjugates described herein include an Fc domain, and Fc monomer, anFc-binding peptide, and albumin protein, or an albumin protein-bindingpeptide covalently linked to one or more dimers of neuraminidaseinhibitors. The dimers of two neuraminidase inhibitors include a firstneuraminidase inhibitor (e.g., a first viral neuraminidase inhibitor offormulas (A-I)-(A-XII)) and a second neuraminidase inhibitor (e.g., asecond viral neuraminidase inhibitor of formulas (A-I)-(A-XII)). Thefirst and second neuraminidase inhibitors are linked to each other byway of a linker, such as a linker described herein. In some embodimentsof the dimers of neuraminidase inhibitors, the first and secondneuraminidase inhibitors are the same. In some embodiments, the firstand second neuraminidase inhibitors are different.

Dimers of neuraminidase inhibitors include homo-dimers of zanamivir oranalogs thereof (e.g., (A-I), (A-II), (A-VI), (A-VII), (A-VIII), or(A-IX)). For example, neuraminidase inhibitor dimers of the inventioninclude dimers having the structure A₁-L-A₂, wherein each A₁ and each A₂is selected from (A-I), (A-II), (A-VI), (A-VII), (A-VIII), and (A-IX).

Dimers of neuraminidase inhibitors include homo-dimers of peramivir oranalogs thereof (e.g., (A-III), (A-IV), or (A-V)). For example,neuraminidase inhibitor dimers of the invention include dimers havingthe structure A₁-L-A₂, wherein each A₁ and each A₂ is selected from(A-III), (A-IV), and (A-V). Dimers of neuraminidase inhibitors includehetero-dimers including zanamivir or analogs thereof and peramivir ofanalogs thereof (e.g., (A-I)-(A-IX)). For example, neuraminidaseinhibitor dimers of the invention include dimers having the structureA₁-L-A₂, wherein each A₁ is selected from (A-I), (A-II), (A-VI),(A-VII), (A-VIII), and (A-IX), and each A₂ is selected from (A-III),(A-IV), and (A-V).

In some embodiments, when T is greater than 1 (e.g., T is 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), each A₁-L-A₂may be independently selected (e.g., independently selected from any ofthe A₁-L-A₂ structures described herein). In some embodiments, E may beconjugated to 2, 3, 4, 5, 6, 7, 8, 9, 10, or more different A₁-L-A₂moieties. In some embodiments, E is conjugated to a first A₁-L-A₂moiety, and a second A₁-L-A₂, moiety. In some embodiments, A₁ and A₂ ofthe first A₁-L-A₂ moiety are independently selected from any one offormulas (A-III)-(A-V):

and A₁ and A₂ of the second A₁-L-A₂ moiety are independently selectedfrom any one of formulas (A-I), (A-II), (A-VI), (A-VII), (A-VIII), or(A-IX):

In some embodiments, the first A₁-L-A₂ moiety is conjugated specificallyto lysine residues of E (e.g., the nitrogen atoms of surface exposedlysine residues of E), and the second A₁-L-A₂ moiety is conjugatedspecifically to cysteine residues of E (e.g., the sulfur atoms ofsurface exposed cysteine residues of E). In some embodiments, the firstA₁-L-A₂ moiety is conjugated specifically to cysteine residues of E(e.g., the sulfur atoms of surface exposed cysteine residues of E), andthe second A₁-L-A₂ moiety is conjugated specifically to lysine residuesof E (e.g., the nitrogen atoms of surface exposed lysine residues of E).

In some embodiments, the disclosure provides a conjugate, or apharmaceutically acceptable salt thereof, described by the formulaebelow:

or a pharmaceutically acceptable salt thereof.

In the conjugates described herein, the squiggly line connected to Eindicates that one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, or 20) dimers of neuraminidase inhibitorsmay be attached to an Fc domain monomer, Fc domain, Fc-binding peptide,albumin protein, or albumin protein-binding peptide. In someembodiments, when n is 1, one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9,or 10) dimers of neuraminidase inhibitors may be attached to an Fcdomain monomer, Fc domain, Fc-binding peptide, albumin protein, oralbumin protein-binding peptide. In some embodiments, when n is 2, oneor more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, or 20) dimers of neuraminidase inhibitors may be attached toan Fc domain. The squiggly line in the conjugates described herein isnot to be construed as a single bond between one or more dimers ofneuraminidase inhibitors and an atom in the Fc domain or albuminprotein. In some embodiments, when T is 1, one dimer of neuraminidaseinhibitors may be attached to an atom in the Fc domain monomer, Fcdomain, Fc-binding peptide, albumin protein, or albumin protein-bindingpeptide. In some embodiments, when T is 2, two dimers of neuraminidaseinhibitors may be attached to an atom in the Fc domain monomer, Fcdomain, Fc-binding peptide, albumin protein, or albumin protein-bindingpeptide.

As described further herein, a linker in a conjugate described herein(e.g., L or L′) may be a branched structure. As described furtherherein, a linker in a conjugate described herein (e.g., L or L′) may bea multivalent structure, e.g., a divalent or trivalent structure havingtwo or three arms, respectively. In some embodiments when the linker hasthree arms, two of the arms may be attached to the first and secondneuraminidase inhibitors and the third arm may be attached to the Fcdomain monomer, Fc domain, Fc-binding peptide, albumin protein, oralbumin protein-binding peptide.

In conjugates having an Fc domain covalently linked to one or moredimers of neuraminidase inhibitors, as represented by the formulaeabove, when n is 2, two Fc domain monomers (each Fc domain monomer isrepresented by E) dimerize to form an Fc domain.

Conjugates of Monomers of Neuraminidase Inhibitors Linked to an FcDomain or an Albumin Protein

In some embodiments, the conjugates described herein include an Fcdomain monomer, Fc domain, Fc-binding peptide, albumin protein, oralbumin protein-binding peptide covalently linked to one or moremonomers of neuraminidase inhibitors. Conjugates of an Fc domain monomeror albumin protein and one or more monomers of neuraminidase inhibitorsmay be formed by linking the Fc domain or albumin protein to each of themonomers of neuraminidase inhibitors through a linker, such as any ofthe linkers described herein.

In the conjugates having an Fc domain or albumin protein covalentlylinked to one or more monomers of neuraminidase inhibitors describedherein, the squiggly line connected to E indicates that one or more(e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, or 20) monomers of neuraminidase inhibitors may be attached to an Fcdomain monomer, Fc domain, Fc-binding peptide, albumin protein, oralbumin protein-binding peptide. In some embodiments, when n is 1, oneor more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) monomers ofneuraminidase inhibitors may be attached to an Fc domain monomer or analbumin protein. In some embodiments, when n is 2, one or more (e.g., 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20)monomers of neuraminidase inhibitors may be attached to an Fc domain.The squiggly line in the conjugates described herein is not to beconstrued as a single bond between one or more monomers of neuraminidaseinhibitors and an atom in the Fc domain monomer, Fc domain, Fc-bindingpeptide, albumin protein, or albumin protein-binding peptide. In someembodiments, when T is 1, one monomer of neuraminidase inhibitor may beattached to an atom in the Fc domain monomer, Fc domain, Fc-bindingpeptide, albumin protein, or albumin protein-binding peptide. In someembodiments, when T is 2, two monomers of neuraminidase inhibitors maybe attached to an atom in the Fc domain monomer, Fc domain, Fc-bindingpeptide, albumin protein, or albumin protein-binding peptide.

In some embodiments, when T is greater than 1 (e.g., T is 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), each A₁-L maybe independently selected (e.g., independently selected from any of theA₁-L structures described herein). In some embodiments, E may beconjugated to 2, 3, 4, 5, 6, 7, 8, 9, 10, or more different A₁-Lmoieties. In some embodiments, E is conjugated to a first A₁-L moiety,and a second A₁-L, moiety. In some embodiments, A₁ of the first A₁-Lmoiety is selected from any one of formulas (A-III)-(A-V):

and A₁ of the second A₁-L moiety is selected from any one of formulas(A-I), (A-II), (A-VI), (A-VII), (A-VIII), or (A-IX):

In some embodiments, the first A₁-L moiety is conjugated specifically tolysine residues of E (e.g., the nitrogen atoms of surface exposed lysineresidues of E), and the second A₁-L moiety is conjugated specifically tocysteine residues of E (e.g., the sulfur atoms of surface exposedcysteine residues of E). In some embodiments, the first A₁-L moiety isconjugated specifically to cysteine residues of E (e.g., the sulfuratoms of surface exposed cysteine residues of E), and the second A₁-Lmoiety is conjugated specifically to lysine residues of E (e.g., thenitrogen atoms of surface exposed lysine residues of E).

As described further herein, a linker in a conjugate having an Fc domainmonomer, Fc domain, Fc-binding peptide, albumin protein, or albuminprotein-binding peptide covalently linked to one or more monomers ofneuraminidase inhibitors described herein (e.g., L or L′) may be adivalent structure having two arms. One arm in a divalent linker may beattached to the monomer of neuraminidase inhibitor and the other arm maybe attached to the Fc domain monomer, Fc domain, Fc-binding peptide,albumin protein, or albumin protein-binding peptide.

In some embodiments, a conjugate containing an Fc domain monomer, Fcdomain, Fc-binding peptide, albumin protein, or albumin protein-bindingpeptide covalently linked to one or more monomers of neuraminidaseinhibitors provided herein is described by any one of formulae below:

or a pharmaceutically acceptable salt thereof.

In conjugates having an Fc domain covalently linked to one or moremonomers of neuraminidase inhibitors, as represented by the formulaeabove, when n is 2, two Fc domain monomers (each Fc domain monomer isrepresented by E) dimerize to form an Fc domain.

Regioisomers of Conjugates Including Zanamivir or Analogs Thereof

Conjugates (e.g., monomer or dimer conjugates as described in detailherein) may be produced as a mixture or regioisomers. A particularregioisomer or mixture of regioisomers may be preferred for reasons suchas ease of synthesis, thermostability, oxidative stability,pharmacokinetics (e.g., metabolic stability or bioavailability),effector binding, or therapeutic efficacy.

In some embodiments, a conjugate of the invention includes zanamivir oran analog thereof (e.g., any of (A-I), (A-II), (A-VI), (A-VII),(A-VIII), (A-IX), or (A-X)). Zanamivir or an analog thereof may beconjugated to an Fc domain or an albumin protein (e.g., by way of alinker) through, for example, the C7 position (see, e.g., (A-I), (A-II),or (A-X)) or through the C9 position (see, e.g., (A-VI) or (A-VII)):

The present disclosure includes a population of monomeric conjugates(e.g., a population of conjugates of formula (M-I)) wherein thepopulation of conjugates includes any of the monomeric conjugatesdescribed herein and one or more of its corresponding regioisomers. Forexample, a population of conjugates may include (1) zanamivir or ananalog thereof conjugated (e.g., by way of a linker) at the C7 positionto an Fc domain or an albumin protein, and (2) zanamivir or an analogthereof conjugated (e.g., by way of a linker) at the C9 position to anFc domain or an albumin protein.

The population of monomeric conjugates may have a specified ratio ofC7-linked conjugate to C9-linked conjugate. For example, the populationof conjugates may have substantially 100% C-7 linked conjugate andsubstantially 0% C-9 linked conjugate. The population of conjugates mayhave about 95% C-7 linked conjugate and about 5% C-9 linked conjugate.The population of conjugates may have about 90% C-7 linked conjugate andabout 10% C-9 linked conjugate. The population of conjugates may haveabout 85% C-7 linked conjugate and about 15% C-9 linked conjugate. Thepopulation of conjugates may have about 80% C-7 linked conjugate andabout 20% C-9 linked conjugate. The population of conjugates may haveabout 75% C-7 linked conjugate and about 25% C-9 linked conjugate. Thepopulation of conjugates may have about 70% C-7 linked conjugate andabout 30% C-9 linked conjugate. The population of conjugates may haveabout 65% C-7 linked conjugate and about 35% C-9 linked conjugate. Thepopulation of conjugates may have about 60% C-7 linked conjugate andabout 40% C-9 linked conjugate. The population of conjugates may haveabout 55% C-7 linked conjugate and about 45% C-9 linked conjugate. Thepopulation of conjugates may have about 50% C-7 linked conjugate andabout 50% C-9 linked conjugate. The population of conjugates may haveabout 45% C-7 linked conjugate and about 55% C-9 linked conjugate. Thepopulation of conjugates may have about 40% C-7 linked conjugate andabout 60% C-9 linked conjugate. The population of conjugates may haveabout 35% C-7 linked conjugate and about 65% C-9 linked conjugate. Thepopulation of conjugates may have about 30% C-7 linked conjugate andabout 70% C-9 linked conjugate. The population of conjugates may haveabout 25% C-7 linked conjugate and about 75% C-9 linked conjugate. Thepopulation of conjugates may have about 20% C-7 linked conjugate andabout 80% C-9 linked conjugate. The population of conjugates may haveabout 15% C-7 linked conjugate and about 85% C-9 linked conjugate. Thepopulation of conjugates may have about 10% C-7 linked conjugate andabout 90% C-9 linked conjugate. The population of conjugates may havesubstantially 0% C-7 linked conjugate and substantially 100% C-9 linkedconjugate.

The population of conjugates may have greater than 99%, 98%, 97%, 96%,95%, 90%, 85%, 80%, 75%, 70%, 60%, 65%, 60%, 55%, or 50% C7-linkedconjugate.

The population of conjugates may have less than 50%, 40%, 30%, 25%, 20%,15%, 10%, 5%, 4%, 3%, 2%, or 1% C9-linked conjugate.

The present disclosure also includes a population of dimeric conjugates(e.g., a population of conjugates of formula (D-I)) wherein thepopulation of conjugates includes any of the dimeric conjugatesdescribed herein and one or more of its corresponding regioisomers. Forexample, a population of conjugates may include a (1) a C7-C7 dimer(e.g., both zanamivir or analog thereof moieties of the dimer areconjugated (e.g., by way of a linker) at their respective C7 positionsto an Fc domain or an albumin protein), (2) a C9-C9 dimer (e.g., bothzanamivir or analog thereof moieties of the dimer are conjugated (e.g.,by way of a linker) at their respective C9 positions to an Fc domain oran albumin protein), and/or (3) a C7-C7 dimer (e.g., one zanamivir oranalog thereof moiety is conjugated (e.g., by way of a linker) to and Fcdomain or an albumin protein through its C7 position and the otherzanamivir or analog thereof moiety is conjugated (e.g., by way of alinker) to an Fc domain or an albumin protein through its C9 position).

The population of dimeric conjugates may have a specified ratio of C7-C7linked conjugate to C7-C9 linked conjugate to C9-C9 linked conjugate.For example, the population of conjugates may have substantially 100%C7-C7 linked conjugate, and substantially 0% C7-C9 or C9-C9 linkedconjugate. The population of conjugates may have substantially 100%C9-C9 linked conjugate, and substantially 0% C7-C7 or C7-C9 linkedconjugate. The population of conjugates may have substantially 100%C7-C9 linked conjugate, and substantially 0% C7-C7 or C9-C9 linkedconjugate.

The population of conjugates may have greater than 99%, 98%, 97%, 96%,95%, 90%, 85%, 80%, 75%, 70%, 60%, 65%, 60%, 55%, or 50% C7-C7 linkedconjugate.

The population of conjugates may have less than 50%, 40%, 30%, 25%, 20%,15%, 10%, 5%, 4%, 3%, 2%, or 1% C9-C9 linked conjugate.

The population of conjugates may have less than 50%, 40%, 30%, 25%, 20%,15%, 10%, 5%, 4%, 3%, 2%, or 1% C7-C9 linked conjugate.

For any of the above-described populations of regioisomers, A₁ and/or A₂(e.g., of (M-I) or (D-I)) may be selected from zanamivir or any of thezanamivir analogs described herein (e.g., any of (A-I), (A-II), (A-VI),(A-VII), (A-VIII), (A-IX), or (A-X)). In particular, the C7-linkedzanamivir or analogs thereof is described by (A-I), (A-II), and (A-X),and C9-linked zanamivir or analogs thereof is described by (A-VI) or(A-VII). Exemplary methods for preparing regioisomers, e.g., C7, C9,C7-C7, C7-C9, and C9-C9 linked regioisomers, are described in Examples100-103, 123 and 124. In some instances, it may be preferable to have95% or more, 96% or more, 97% or more, 98% or more, 99% or more, orsubstantially 100% C7 linked monomer conjugates or C7-C7 linked dimerconjugates. In these instances, it may be preferable to prepare theintermediate with a method that forms substantially C7 linked monomer orC7-C7 linked dimer intermediates, such as the methods described, forexample, in Examples 103 and 123. The method of Example 103 is exemplaryof methods used to achieve primarily the C7 or C7-C7 linked intermediateand may be used to prepare any intermediate described herein.

In preferred embodiments, the conjugate is a conjugate of any one offormulas (D-I)-(D-X), (D′-I), (M-I)-(M-X), or (M′-I), wherein A₁ and/orA₂ are described by formula (A-I), (A-II), or (A-X) and Y is

wherein R₇ is selected from H, C1-C20 alkyl, C3-C20 cycloalkyl, C3-C20heterocycloalkyl; C5-C15 aryl, and C2-C15 heteroaryl. In preferredembodiments, A₁ and/or A₂ are described by formula (A-I) (e.g.,zanamivir). In preferred embodiments, R₇ is C1-C20 alkyl (e.g., —CH₃,—CH₂CH₃, —CH₂CH₂CH₃). Such conjugates have been shown to exhibitincreased stability of the C7-linkage, resulting in less C7 to C9migration. The resulting product is therefore expected to be morehomogenous and exhibit increased efficacy.

III. Fc Domain Monomers and Fc Domains

An Fc domain monomer includes a hinge domain, a CH₂ antibody constantdomain, and a CH₃ antibody constant domain. The Fc domain monomer can beof immunoglobulin antibody isotype IgG, IgE, IgM, IgA, or IgD. The Fcdomain monomer can also be of any immunoglobulin antibody isotype (e.g.,IgG1, IgG2a, IgG2b, IgG3, or IgG4). The Fc domain monomer can be of anyimmunoglobulin antibody allotype (e.g., IGHG1*01 (i.e., G1m(za)),IGHG1*07 (i.e., G1m(zax)), IGHG1*04 (i.e., G1m(zav)), IGHG1*03 (G1m(f)),IGHG1*08 (i.e., G1m(fa)), IGHG2*01, IGHG2*06, IGHG2*02, IGHG3*01,IGHG3*05, IGHG3*10, IGHG3*04, IGHG3*09, IGHG3*11, IGHG3*12, IGHG3*06,IGHG3*07, IGHG3*08, IGHG3*13, IGHG3*03, IGHG3*14, IGHG3*15, IGHG3*16,IGHG3*17, IGHG3*18, IGHG3*19, IGHG2*04, IGHG4*01, IGHG4*03, or IGHG4*02)(as described in, for example, in Vidarsson et al. IgG subclasses andallotypes: from structure to effector function. Frontiers in Immunology.5(520): 1-17 (2014)). The Fc domain monomer can also be of any species,e.g., human, murine, or mouse. A dimer of Fc domain monomers is an Fcdomain that can bind to an Fc receptor, which is a receptor located onthe surface of leukocytes.

In some embodiments, an Fc domain monomer in the conjugates describedherein may contain one or more amino acid substitutions, additions,and/or deletion relative to an Fc domain monomer having a sequence ofany one of SEQ ID NOs: 1-68. In some embodiments, an Asn in an Fc domainmonomer in the conjugates as described herein may be replaced by Ala inorder to prevent N-linked glycosylation (see, e.g., SEQ ID NOs: 12-15,where Asn to Ala substitution is labeled with *). In some embodiments,an Fc domain monomer in the conjugates described herein may alsocontaining additional Cys additions (see, e.g., SEQ ID NOs: 9, 10, and11, where Cys additions are labeled with *).

In some embodiments, an Fc domain monomer in the conjugates as describedherein includes an additional moiety, e.g., an albumin-binding peptide,a purification peptide (e.g., a hexa-histidine peptide (HHHHHH (SEQ IDNO: 72)), or a signal sequence (e.g., IL2 signal sequenceMYRMQLLSCIALSLALVTNS (SEQ ID NO: 73)) attached to the N- or C-terminusof the Fc domain monomer. In some embodiments, an Fc domain monomer inthe conjugate does not contain any type of antibody variable region,e.g., VH, VL, a complementarity determining region (CDR), or ahypervariable region (HVR).

In some embodiments, an Fc domain monomer in the conjugates as describedherein may have a sequence that is at least 95% identical (e.g., 97%,99%, or 99.5% identical) to the sequence of any one of SEQ ID NOs: 1-68shown below. In some embodiments, an Fc domain monomer in the conjugatesas described herein may have a sequence of any one of SEQ ID NOs: 1-68shown below.

SEQ ID NO: 1: murine Fc-IgG2a with IL2 signal sequence atthe N-terminus (bold)MYRMQLLSCIALSLALVTNSPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEVVTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGKSEQ ID NO: 2: mature murine Fc-IgG2aPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEVVTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGKSEQ ID NO: 3: human Fc-IgG1 with IL2 signal sequence atthe N-terminus (bold) and N-terminal MVRS amino acidresidues added (underlined) MYRMQLLSCIALSLALVTNSMVRSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKSEQ ID NO: 4: mature human Fc-IgG1 with N-terminal MVRSamino acid residues added (underlined)MVRSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKSEQ ID NO: 5: murine Fc-IgG2a with IL2 signal sequence(bold) at the N-terminus and hexa-histidine peptide(italicized) at the C-terminusMYRMQLLSCIALSLALVTNSPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEVVTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGKHHHHHHSEQ ID NO: 6: mature murine Fc-IgG2a with hexa-histidinepeptide (italicized) at the C-terminusPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEVVTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGKHHHHHHSEQ ID NO: 7: human Fc-IgG1 with IL2 signal sequence (bold)at the N-terminus, N-terminal MVRS amino acid residues added(underlined), and hexa-histidine peptide (italicized) at the C-terminusMYRMQLLSCIALSLALVTNS MVRSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKHHHHHHSEQ ID NO: 8: mature human Fc-IgG1 with hexa-histidinepeptide (italicized) at the C-terminus and N-terminalMVRS amino acid residues added (underlined)MVRSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKHHHHHHSEQ ID NO: 9: human Fc-IgG1 with IL2 signal sequence (bold) atthe N-terminus, N-terminal MVRS amino acid residues added(underlined), two additional cysteines in the hinge region (*),and hexa-histidine peptide (italicized) at the C-terminusMYRMQLLSCIALSLALVTNS MVRSDKTHTCPPCPPC*KC*PAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKHHHHHHSEQ ID NO: 10: mature human Fc-IgG1 with N-terminal MVRS aminoacid residues added (underlined), two additional cysteines inthe hinge region (*), and hexa-histidine peptide (italicized)at the C-terminusMVRSDKTHTCPPCPPC*KC*PAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKHHHHHHSEQ ID NO: 11: mature human Fc-IgG1 with N-terminal MVRS aminoacid residues added (underlined) and two additional cysteinesin the hinge region (*)MVRSDKTHTCPPCPPC*KC*PAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKSEQ ID NO: 12: murine Fc-IgG2a with IL2 signal sequence (bold)at the N-terminus, Asn to Ala substitution (*), and hexa-histidine peptide (italicized) at the C-terminusMYRMQLLSCIALSLALVTNSPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYA*STLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEVVTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGKHHHHHHSEQ ID NO: 13: mature murine Fc-IgG2a with Asn to Ala substitution(*) and hexa-histidine peptide (italicized) at the C-terminusPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYA*STLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGKHHHHHHSEQ ID NO: 14: human Fc-IgG1 with IL2 signal sequence (bold) atthe N-terminus, N-terminal MVRS amino acid residues added(underlined), Asn to Ala substitution (*), and hexa-histidinepeptide (italicized) at the C-terminus MYRMQLLSCIALSLALVTNSMVRSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYA*STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKHHHHHHSEQ ID NO: 15: mature human Fc-IgG1 with Asn to Ala substitution(*), N-terminal MVRS amino acid residues added (underlined), andhexa-histidine peptide (italicized) at the C-terminusMVRSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYA*STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKHHHHHHSEQ ID NO: 16: human IgG1 Fc with Human Serum Albumin SignalSequence (bold) at the N-terminus and N-terminal ISAMVRS aminoacid residues added (underlined) MKWVTFISLLFLFSSAYSISAMVRSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKITSKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKSEQ ID NO: 17: human IgG1 Fc with Human Serum Albumin SignalSequence (bold) at the N-terminus, N-terminal ISAMVRS aminoacid residues added (underlined), C-terminal G45 linker(italicized), and C-terminal c-Myc tag (underlined, italicized)MKWVTFISLLFLFSSAYS ISAMVRSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGS EQKLISEEDLSEQ ID NO: 18: mature human IgG1 Fc with N-terminal ISAMVRSamino acid residues added (underlined), C-terminal G45 linker(italicized), and C-terminal c-Myc tag (underlined, italicized)ISAMVRSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGS EQKLISEEDLSEQ ID NO: 19: human IgG1 Fc with Human Serum Albumin SignalSequence (bold), N-terminal ISAMVRS amino acid residues added(underlined), and lysine to serine modification (*) to preventlysine conjugation at this site MKWVTFISLLFLFSSAYSISAMVRSDKTHTCPPCPAPELLGGPSVFLFPPKPS*DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKSEQ ID NO: 20: mature human IgG1 Fc with N-terminal ISAMVRSamino acid residues added (underlined) and lysine to serinemodification (*) to prevent lysine conjugation at this siteISAMVRSDKTHTCPPCPAPELLGGPSVFLFPPKPS*DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKSEQ ID NO: 21: human IgG1 Fc with Human Serum Albumin SignalSequence (bold) at the N-terminus, N-terminal ISAMVRS aminoacid residues added (underlined), lysine to serine modification(*) to prevent lysine conjugation at this site, C-terminal G4Slinker (italicized), and C-terminal C-Myc tag (underlined, italicized)MKWVTFISLLFLFSSAYS ISAMVRSDKTHTCPPCPAPELLGGPSVFLFPPKPS(*)DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSEQKLISEEDLSEQ ID NO: 22: mature human IgG1 Fc with N-terminal ISAMVRSamino acid residues added (underlined), lysine to serinemodification (*) to prevent lysine conjugation at this site,C-terminal G45 linker (italicized), and C-terminal C-Myc tag(underlined, italicized)ISAMVRSDKTHTCPPCPAPELLGGPSVFLFPPKPS(*)DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGS EQKLISEEDLSEQ ID NO: 23: human IgG1 Fc with Human Serum Albumin SignalSequence (bold) at the N-terminus, N-terminal ISAMVRS aminoacid residues added (underlined), Asn to Ala substitution(*), C-terminal G45 linker (italicized), and C-terminalC-myc tag (underlined, italicized) MKWVTFISLLFLFSSAYSISAMVRSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYA(*)STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGS EQKLISEEDLSEQ ID NO: 24: mature human IgG1 Fc with N-terminal ISAMVRSamino acid residues added (underlined), Asn to Ala substitution(*), C-terminal G45 linker (italicized), and C-terminal C-myctag (underlined, italicized)ISAMVRSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYA(*)STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGS EQKLISEEDLSEQ ID NO: 25: human IgG1 Fc with Human Serum Albumin SignalSequence (bold) at the N-terminus, N-terminal ISAMVRS aminoacid residues added (underlined), H310A (*) and H435A (*)mutations to impede FcRn binding, C-terminal G45 (italicized),and C-terminal C-myc tag (underlined, italicized) MKWVTFISLLFLFSSAYSISAMVRSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLA(*)QDWLNGKEYKCKVSNKALPAPIEKTISKA(*)KGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNAYTQKSLSLSPGGGGGS EQKLISEEDLSEQ ID NO: 26: mature human IgG1 Fc with Human Serum AlbuminSignal Sequence (bold) at the N-terminus, N-terminal ISAMVRSamino acid residues added (underlined), with H310A (*) andH435A (*) mutations to impede FcRn binding, C-terminal G45(italicized), and C-terminal C-myc tag (underlined, italicized)ISAMVRSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLA(*)QDWLNGKEYKCKVSNKALPAPIEKTISKA(*)KGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNAYTQKSLSLSPGGGGGS EQKLISEEDLSEQ ID NO: 27: human IgG1 Fc with Human Serum Albumin SignalSequence (bold) at the N-terminus, N-terminal ISAMVRS aminoacid residues added (underlined), C-terminal G45 linker(italicized), and C-terminal mutated (lysine to phenylalanine,bold) C-myc tag (underlined, italicized) MKWVTFISLLFLFSSAYSISAMVRSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGS EQ

LISEEDL SEQ ID NO: 28: mature human IgG1 Fc with N-terminal ISAMVRSamino acid residues added (underlined), C-terminal G45 linker(italicized), and C-terminal mutated (lysine to phenylalanine,bold) C-myc tag (underlined, italicized)ISAMVRSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGS EQ

LISEEDL SEQ ID NO: 29: human IgG1 Fc with Human Serum Albumin SignalSequence (bold) at the N-terminus, N-terminal ISAMVRS aminoacid residues added (underlined), Asn to Ala substitution (*),C-terminal G45 linker (italicized), and C-terminal mutated(lysine to phenylalanine, bold) C-myc tag (underlined, italicized)MKWVTFISLLFLFSSAYS ISAMVRSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYA(*)STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSEQFLISEEDLSEQ ID NO: 30: mature human IgG1 Fc with N-terminalMVRS amino acid residues added (underlined), Asn toAla substitution (*), C-terminal G45 linker (italicized),and C-terminal mutated (lysine to phenylalanine, bold)C-myc tag (underlined, italicized)ISAMVRSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYA(*)STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGS EQ

LISEEDL SEQ ID NO: 31: human IgG1 Fc with Human Serum AlbuminSignal Sequence (bold) at the N-terminus, allotype G1m(fa)(bold italics), C-termainl G45 linker (italicized), andC-terminal mutated (lysine to phenylalanine, bold) C-myctag (underlined)MKWVTFISLLFLFSSAYSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR

E

TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSEQFLISEEDLSEQ ID NO: 32: human IgG1 Fc with Human Serum Albumin SignalSequence (bold) at the N-terminus, allotype G1m(fa) (bold italics)MKWVTFISLLFLFSSAYSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR

E

TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKSEQ ID NO: 33: mature human IgG1 Fc with a YTE triple mutation(bold and underlined) with N-terminal MVRS amino acid residuesadded (underlined) MVRSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTL Y I T R EPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKSEQ ID NO: 34: human IgG1 Fc with Human Serum Albumin SignalSequence (bold) at the N-terminus, contains residues EPKSScomprising the full hinge region on the N-terminus of maturehuman IgG1 Fc (underlined), Cys to Ser substitution (#),allotype G1m(fa) (bold italics) MKWVTFISLLFLFSSAYSEPKSS(#)DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR

E

TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKSEQ ID NO: 35: human IgG1 Fc with murine IgG signal sequence(bold) at the N-terminus, with removal of EPKSSD hinge residuesfrom the N-terminus of the mature human IgG1 Fc, allotype G1m(fa)(bold italics)MGWSCIILFLVATATGVHSKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR

E

TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKSEQ ID NO: 36: mature human IgG1 Fc with a YTE triple mutation(bold and underlined), with removal of EPKSSD hinge residuesfrom the N-terminus of the mature human IgG1 Fc, allotype G1m(fa)(bold italics) KTHTCPPCPAPELLGGPSVFLFPPKPKDTL Y I T R EPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR

E

TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKSEQ ID NO: 37: mature human IgG1 Fc with an LS double mutation(bold and underlined), with removal of EPKSSD hinge residuesfrom the N-terminus of the mature human IgG1 Fc, allotype G1m(fa)(bold italics)KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR

E

TKN QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV LHEA LH S HYTQKSLSLSPGKSEQ ID NO: 38: mature human IgG1 Fc with Human Serum AlbuminSignal Sequence (bold) at the N-terminus, a YTE triple mutation(bold and underlined), allotype G1m(fa) (bold italics), C-terminalG45 linker (italicized), and C-terminal C-myc tag (underlined)MKWVTFISLLFLFSSAYSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTL Y I T R EPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR

E

TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSEQKLISEEDLSEQ ID NO: 39: mature human Fc IgG1, wherein X₁ is Met or Trp,X₂ is Ser or Thr, X₃ is Thr or Glu, X₄ is Asp or Glu, and X₅is Leu or Met, X₆ is Met or Leu, and X₇ is Asn or SerDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLX ₁IX ₂RX₃PEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRX ₄EX ₅TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVX ₆HEALHX ₇HYTQKSLSLSPGSEQ ID NO: 40: mature human Fc IgG1 wherein X₄ is Asp or Glu,and X₅ is Leu or MetDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRX ₄EX ₅TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSEQ ID NO: 41: mature human Fc IgG1 with a YTE triple mutation(bold and underlined), and wherein X₄ is Asp or Glu, and X₅ isLeu or Met DKTHTCPPCPAPELLGGPSVFLFPPKPKDTL Y I T R EPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRX ₄EX ₅TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSEQ ID NO: 42: mature human Fc IgG1 with a YTE triple mutation(bold and underlined), allotype G1m(fa) (bold italics)DKTHTCPPCPAPELLGGPSVFLFPPKPKDTL Y I T R EPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR

E

TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSEQ ID NO: 43: mature human Fc IgG1 with a YTE triple mutation(bold and underlined), allotype G1m(f) (bold italics)DKTHTCPPCPAPELLGGPSVFLFPPKPKDTL Y I T R EPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR

E

TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSEQ ID NO: 44: mature human Fc IgG1 with a LS double mutation(bold and underlined), and wherein X₄ is Asp or Glu, and X₅is Leu or MetDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRX ₄EX ₅TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV LHEALH S HYTQKSLSLSPGSEQ ID NO: 45: mature human Fc IgG1 with a LS double mutation(bold and underlined), allotype G1m(fa) (bold italics)DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR

E

TK NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV LHE ALH S HYTQKSLSLSPGSEQ ID NO: 46: mature human Fc IgG1 with a LS double mutation(bold and underlined), allotype G1m(f) (bold italics)DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR

E

TK NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV LHE ALH S HYTQKSLSLSPGSEQ ID NO: 47: mature human Fc IgG1 with mouse heavy chainMIgG Vh signal sequence (bold), Cys to Ser substitution (#),and wherein X₁ is Met or Trp, X₂ is Ser or Thr, X₃ is Thr orGlu, X₄ is Asp or Glu, and X₅ is Leu or Met, X₆ is Met or Leu,and X₇ is Asn or SerMGWSCIILFLVATATGVHSNVNHKPSNTKVDKKVEPKSS(#)DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLX ₁IX ₂RX₃PEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRX ₄EX ₅TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVX ₆HEALHX ₇HYTQKSLSLSPGSEQ ID NO: 48: mature human IgG1 Fc with mouse heavy chainMIgG Vh signal sequence (bold), Cys to Ser substitution (#),allotype G1m(fa) (bold italics)MGWSCIILFLVATATGVHSNVNHKPSNTKVDKKVEPKSS(#)DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR

E

TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKSEQ ID NO: 49: mature human IgG1 Fc with mouse heavy chain MIgGVh signal sequence (bold), Cys to Ser substitution (#), allotypeG1m(f) (bold italics)MGWSCIILFLVATATGVHSNVNHKPSNTKVDKKVEPKSS(#)DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR

E

TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKSEQ ID NO: 50: mature human IgG1 Fc with mouse heavy chain MIgGVh signal sequence (bold), Cys to Ser substitution (#), M428L, N4345 mutations (Bold/Underlined), allotype G1m(fa) (bold italics)MGWSCIILFLVATATGVHSNVNHKPSNTKVDKKVEPKSS(#)DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR

E

TKNQVSLTCLVKGFYPSDIAVEWESNGQPE NNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV LHEALH S HYTQKSLSLSPGKSEQ ID NO: 51: mature human IgG1 Fc with mouse heavy chain MIgGVh signal sequence (bold), Cys to Ser substitution (#), M428L,N4345 mutations (Bold/Underlined), allotype G1m(f) (bold italics)MGWSCIILFLVATATGVHSNVNHKPSNTKVDKKVEPKSS(#)DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR

E

TKNQVSLTCLVKGFYPSDIAVEWESNGQPE NNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV LHEALH S HYTQKSLSLSPGKSEQ ID NO: 52: mature human IgG1 Fc with mouse heavy chain MIgGVh signal sequence (bold), Cys to Ser substitution (#), YTE triple mutation (bold and underlined), allotype G1m(fa) (bold italics)MGWSCIILFLVATATGVHSNVNHKPSNTKVDKKVEPKSS(#)DKTHTCPPCPAPELLGGPSVFLFPPKPKDTL Y I T R EPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR

E

TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKSEQ ID NO: 53: mature human IgG1 Fc with mouse heavy chain MIgGVh signal sequence (bold), Cys to Ser substitution (#), YTE triplemutation (bold and underlined), allotype G1m(f) (bold italics)MGWSCIILFLVATATGVHSNVNHKPSNTKVDKKVEPKSS(#)DKTHTCPPCPAPELLGGPSVFLFPPKPKDTL Y I T R EPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR

E

TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKSEQ ID NO: 54: mature human IgG1 Fc with mouse heavy chain MIgGVh signal sequence (bold), N-terminal ISAMVRS amino acid residuesadded (italicized), M428L, N4345 mutations (bold/underlined), G45linker (italicized), and C-terminal C-myc-tag (underlined),allotype G1m(f) (bold italics) MGWSCIILFLVATATGVHSISAMVRSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR

E

TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRWQQGNVFSCSV LHEALH S HYTQKSLSLSPGGGGGSEQKLISEEDLSEQ ID NO: 55: mature human IgG1 Fc with mouse heavy chainMIgG Vh signal sequence (bold), N-terminal ISAMVRS aminoacid residues added (italicized), M428L, N4345 mutations(bold/underlined), G45 linker (italicized), C-terminalC-myc-tag (underlined), allotype G1m(fa) (bold italics)MGWSCIILFLVATATGVHS ISAMVRSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR

E

TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRWQQGNVFSCSV LHEALH S HYTQKSLSLSPGGGGGSEQKLISEEDLSEQ ID NO: 56: mature human IgG1 Fc with mouse heavy chainMIgG Vh signal sequence (bold), N-terminal ISAMVRS aminoacid residues added (italicized), YTE triple mutant (bold/underlined), G45 linker (italicized), and C-terminalC-myc-tag (underlined), allotype G1m(f) (bold italics)MGWSCIILFLVATATGVHS ISAMVRSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTL Y I T R EPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR

E

TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSEQKLISEEDLSEQ ID NO: 57: mature human IgG1 Fc with mouse heavy chainMIgG Vh signal sequence (bold), N-terminal ISAMVRS aminoacid residues added (italicized), YTE triple mutant (bold/underlined), G45 linker (italicized), C-terminal C-myc-tag(underlined), allotype G1m(fa) (bold italics) MGWSCIILFLVATATGVHSISAMVRSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTL Y I T R E PEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR

E

TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSEQKLISEEDLSEQ ID NO: 58: mature human IgG1 with mouse heavy chainMIgG1 signal sequence (bold), Cys to Ser substitution (#),C-terminal G45 (italics), and C-terminal IgA peptide(underline), allotype G1m(fa) (bold italics)MGWSCIILFLVATATGVHSEPKSS(#)DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR

E

TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSQRNPRLRLIRRHPTLRIPPISEQ ID NO: 59: mature human IgG1 with mouse heavy chainMIgG1 signal sequence (bold), Cys to Ser substitution (#),M428L, N4345 mutations (bold/underlined), C-terminal G45(italics), and C-terminal IgA peptide (underline), allotypeG1m(fa) (bold italics)MGWSCIILFLVATATGVHSEPKSS(#)DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR

E

TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVLHEALHSHYTQKSLSLSPGGGGGSQRNPRLRLIRRHPTLRIPPISEQ ID NO: 60: mature human Fc IgG1, Z₁ is Cys or Ser, andwherein X₁ is Met or Trp, X₂ is Ser or Thr, X₃ is Thr or Glu,X₄ is Asp or Glu, and X₅ is Leu or Met, X₆ is Met or Leu,and X₇ is Asn or Ser NVNHKPSNTKVDKKVEPKSZ₁DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLX ₁IX ₂RX ₃PEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRX ₄EX ₅TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVX ₆HEALHX ₇HYTQKSLSLSPGKSEQ ID NO: 61: mature human Fc IgG1, Cys to Ser substitution(#), and wherein X₁ is Met or Trp, X₂ is Ser or Thr, X₃ is Thror Glu, X₄ is Asp or Glu, and X₅ is Leu or Met, X₆ is Met orLeu, and X₇ is Asn or SerNVNHKPSNTKVDKKVEPKSS(#)DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLX ₁₁ X ₂RX₃PEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRX ₄EX ₅TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVX ₆HEALHX ₇HYTQKSLSLSPGKSEQ ID NO: 62: mature human IgG1 Fc, Cys to Ser substitution(#), X₄ is Asp or Glu, and X₅ is Leu or MetNVNHKPSNTKVDKKVEPKSS(#)DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRX ₄EX ₅TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKSEQ ID NO: 63: mature human IgG1 Fc, Cys to Ser substitution(#), allotype G1m(f) (bold italics)NVNHKPSNTKVDKKVEPKSS(#)DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR

E

TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKSEQ ID NO: 64: mature human IgG1 Fc, Cys to Ser substitution(#), allotype G1m(fa) (bold italics)NVNHKPSNTKVDKKVEPKSS(#)DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR

E

TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKSEQ ID NO: 65: mature human IgG1 Fc, Cys to Ser substitution(#), M428L, N4345 mutations (Bold/Underlined), allotype G1m(fa)(bold italics)NVNHKPSNTKVDKKVEPKSS(#)DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR

E

TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY SKLTVDKSRWQQGNVFSCSV LHEALH S HYTQKSLSLSPGKSEQ ID NO: 66: mature human IgG1 Fc, Cys to Ser substitution(#), M428L, N4345 mutations (Bold/Underlined), allotype G1m(f)(bold italics)NVNHKPSNTKVDKKVEPKSS(#)DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR

E

TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY SKLTVDKSRWQQGNVFSCSV LHEALH S HYTQKSLSLSPGKSEQ ID NO: 67: mature human IgG1 Fc, Cys to Ser substitution (#),YTE triple mutation (bold and underlined), allotype G1m(fa)(bold italics) NVNHKPSNTKVDKKVEPKSS(#)DKTHTCPPCPAPELLGGPSVFLFPPKPKDTL YI T R E PEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR

E

TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKSEQ ID NO: 68: mature human IgG1 Fc, Cys to Ser substitution(#), YTE triple mutation (bold and underlined), allotype G1m(f)(bold italics) NVNHKPSNTKVDKKVEPKSS(#)DKTHTCPPCPAPELLGGPSVFLFPPKPKDTL YI T R E PEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR

E

TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

As defined herein, an Fc domain includes two Fc domain monomers that aredimerized by the interaction between the C_(H)3 antibody constantdomains, as well as one or more disulfide bonds that form between thehinge domains of the two dimerizing Fc domain monomers. An Fc domainforms the minimum structure that binds to an Fc receptor, e.g., Fc-gammareceptors (i.e., Fcγ receptors (FcγR)), Fc-alpha receptors (i.e., Fcαreceptors (FcαR)), Fc-epsilon receptors (i.e., Feε receptors (FcεR)),and/or the neonatal Fc receptor (FcRn). In some embodiments, an Fcdomain of the present invention binds to an Fcγ receptor (e.g., FcRn,FcγRI (CD64), FcγRIIa (CD32), FcγRIIb (CD32), FcγRIIIa (CD16a), FcγRIIIb(CD16b)), and/or FcγRIV and/or the neonatal Fc receptor (FcRn).

In some embodiments, the Fc domain monomer or Fc domain of the inventionis an aglycosylated Fc domain monomer or Fc domain (e.g., an Fc domainmonomer or and Fc domain that maintains engagement to an Fc receptor(e.g., FcRn). For example, the Fc domain is an aglycosylated IgG1variants that maintains engagement to an Fc receptor (e.g., an IgG1having an amino acid substitution at N297 and/or T299 of theglycosylation motif). Exemplary aglycosylated Fc domains and methods formaking aglycosylated Fc domains are known in the art, for example, asdescribed in Sazinsky S. L. et at, Aglycosylated immunoglobulin G1variants productively engage activating Fc receptors, PNAS, 2008,105(51):20167-20172, which is incorporated herein in its entirety.

In some embodiments, the Fc domain or Fc domain monomer of the inventionis engineered to enhance binding to the neonatal Fc receptor (FcRn). Forexample, the Fc domain may include the triple mutation corresponding toM252Y/S254T/T256E (YTE) (e.g., an IgG1, such as a human or humanizedIgG1 having a YTE mutation, for example SEQ ID NO: 33, SEQ ID NO: 36,SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO:43, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 56, or SEQ ID NO: 57). TheFc domain may include the double mutant corresponding to M428L/N434S(LS) (e.g., an IgG1, such as a human or humanized IgG1 having an LSmutation, such as SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 44, SEQ IDNO: 45, SEQ ID NO: 46, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 54, SEQID NO: 55, or SEQ ID NO: 59). The Fc domain may include the singlemutant corresponding to N434H (e.g., an IgG1, such as a human orhumanized IgG1 having an N434H mutation). The Fc domain may include thesingle mutant corresponding to C220S (e.g., an IgG1, such as a human orhumanized IgG1 having a C220S mutation, such as SEQ ID NOs: 34, SEQ IDNO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQID NO: 52, SEQ ID NO: 53, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 61,SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO:66, SEQ ID NO: 67, SEQ ID NO: 68). The Fc domain may include acombination of one or more of the above-described mutations that enhancebinding to the FcRn. Enhanced binding to the FcRn may increase thehalf-life Fc domain-containing conjugate. For example, incorporation ofone or more amino acid mutations that increase binding to the FcRn(e.g., a YTE mutation, an LS mutation, or an N434H mutation) mayincrease the half life of the conjugate by 5%, 10%, 15%, 20%, 30%, 40%,50%, 60%, 70%, 80%, 90%. 100%, 200%, 300%, 400%, 500% or more relativeto a conjugate having an the corresponding Fc domain without themutation that enhances FcRn binding. Exemplary Fc domains with enhancedbinding to the FcRN and methods for making Fc domains having enhancedbinding to the FcRN are known in the art, for example, as described inMaeda, A. et at, Identification of human IgG1 variant with enhanced FcRnbinding and without increased binding to rheumatoid factor autoantibody,MABS, 2017, 9(5):844-853, which is incorporated herein in its entirety.As used herein, an amino acid “corresponding to” a particular amino acidresidue (e.g., of a particular SEQ ID NO.) should be understood toinclude any amino acid residue that one of skill in the art wouldunderstand to align to the particular residue (e.g., of the particularsequence). For example, anyone of SEQ ID NOs: 1-68 may be mutated toinclude a YTE mutation, an LS mutation, and/or an N434H mutation bymutating the “corresponding residues” of the amino acid sequence.

As used herein, a sulfur atom “corresponding to” a particular cysteineresidue of a particular SEQ ID NO. should be understood to include thesulfur atom of any cysteine residue that one of skill in the art wouldunderstand to align to the particular cysteine of the particularsequence. The protein sequence alignment of human IgG1 (UniProtKB:P01857; SEQ ID NO: 94), human IgG2 (UniProtKB: P01859; SEQ ID NO: 95),human IgG3 (UniProtKB: P01860; SEQ ID NO: 96), and human IgG4(UniProtKB: P01861; SEQ ID NO: 97) is provided below (aligned withClustal Omega Multiple Pairwise Alignment). The alignment indicatescysteine residues (e.g., sulfur atoms of cysteine residues) that“correspond to” one another (in boxes and indicated by the • symbol).One of skill in the art would readily be able to perform such analignment with any IgG variant of the invention to determine the sulfuratom of a cysteine that corresponds to any sulfur atom of a particularcysteine of a particular SEQ ID NO. described herein (e.g., any one ofSEQ ID NOs: 1-68). For example, one of skill in the art would readily beable to determine that Cys10 of SEQ ID NO: 10 (the first cysteine of theconserved CPPC motif of the hinge region of the Fc domain) correspondsto, for example, Cys109 of IgG1, Cys106 of IgG2, Cys156 of IgG3, Cys29of SEQ ID NO: 1, Cys9 of SEQ ID NO: 2, Cys30 of SEQ ID NO: 3, or Cys10of SEQ ID NO: 10.

In some embodiments, the Fc domain or Fc domain monomer of the inventionhas the sequence of any one of SEQ ID NOs: 39-68 may further includeadditional amino acids at the N-terminus (Xaa)x and/or additional aminoacids at the C-terminus (Xaa)z, wherein Xaa is any amino acid and x andz are a whole number greater than or equal to zero, generally less than100, preferably less than 10 and more preferably 0, 1, 2, 3, 4, or 5. Insome embodiments, the additional amino acids are least 70% (e.g., 70%,75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to oneor more consecutive amino acids of SEQ ID NO: 94. For example, theadditional amino acids may be a single amino aid on the C-terminuscorresponding to Lys330 of IgG1 (SEQ ID NO: 94).

As used herein, a nitrogen atom “corresponding to” a particular lysineresidue of a particular SEQ ID NO. should be understood to include thenitrogen atom of any lysine residue that one of skill in the art wouldunderstand to align to the particular lysine of the particular sequence.The protein sequence alignment of human IgG1 (UniProtKB: P01857; SEQ IDNO: 94), human IgG2 (UniProtKB: P01859; SEQ ID NO: 95), human IgG3(UniProtKB: P01860; SEQ ID NO: 96), and human IgG4 (UniProtKB: P01861;SEQ ID NO: 97) is provided below (aligned with Clustal Omega MultiplePairwise Alignment). The alignment indicates lysine residues (e.g.,nitrogen atoms of lysine residues) that “correspond to” one another (inboxes and indicated by the * symbol). One of skill in the art wouldreadily be able to perform such an alignment with any IgG variant of theinvention to determine the nitrogen atom of a lysine that corresponds toany nitrogen atom of a particular lysine of a particular SEQ ID NO.described herein (e.g., any one of SEQ ID NOs: 1-68). For example, oneof skill in the art would readily be able to determine that Lys35 of SEQID NO: 10 corresponds to, for example, Lys129 of IgG1, Lys126 of IgG2,Lys176 of IgG3, Lys51 of SEQ ID NO: 1, Lys31 of SEQ ID NO: 2, Lys50 ofSEQ ID NO: 3, or Lys30 of SEQ ID NO: 10.

Protein sequence alignment of IgG1 (SEQ ID NO: 94), IgG2 (SEQ IDNO: 95), IgG3 (SEQ ID NO: 96), and IgG4 (SEQ ID NO: 97)

Activation of Immune Cells

Fc-gamma receptors (FcγRs) bind the Fc portion of immunoglobulin G (IgG)and play important roles in immune activation and regulation. Forexample, the IgG Fc domains in immune complexes (ICs) engage FcγRs withhigh avidity, thus triggering signaling cascades that regulate immunecell activation. The human FcγR family contains several activatingreceptors (FcγRI, FcγRIIa, FcγRIIc, FcγRIIIa, and FcγRIIIb) and oneinhibitory receptor (FcγRIIb). FcγR signaling is mediated byintracellular domains that contain immune tyrosine activating motifs(ITAMs) for activating FcγRs and immune tyrosine inhibitory motifs(ITIM) for inhibitory receptor FcγRIIb. In some embodiments, FcγRbinding by Fc domains results in ITAM phosphorylation by Src familykinases; this activates Syk family kinases and induces downstreamsignaling networks, which include PI3K and Ras pathways.

In the conjugates described herein, the portion of the conjugatesincluding monomers or dimers of neuraminidase inhibitors bind to andinhibits viral neuraminidase leading to inhibition of viral replication,while the Fc domain portion of the conjugates bind to FcγRs (e.g., FcRn,FcγRI, FcγRIIa, FcγRIIc, FcγRIIIa, and FcγRIIIb) on immune cells andactivate phagocytosis and effector functions, such as antibody-dependentcell-mediated cytotoxicity (ADCC), thus leading to the engulfment anddestruction of viral particles by immune cells and further enhancing theantiviral activity of the conjugates. Examples of immune cells that maybe activated by the conjugates described herein include, but are notlimited to, macrophages, neutrophils, eosinophils, basophils,lymphocytes, follicular dendritic cells, natural killer cells, and mastcells.

IV. Albumin Proteins and Albumin Protein-Binding Peptides AlbuminProteins

An albumin protein of the invention may be a naturally-occurring albuminor a variant thereof, such as an engineered variant of anaturally-occurring albumin protein. Variants include polymorphisms,fragments such as domains and sub-domains, and fusion proteins. Analbumin protein may include the sequence of an albumin protein obtainedfrom any source. Preferably the source is mammalian, such as human orbovine. Most preferably, the albumin protein is human serum albumin(HSA), or a variant thereof. Human serum albumins includes any albuminprotein having an amino acid sequence naturally occurring in humans, andvariants thereof. An albumin protein coding sequence is obtainable bymethods know to those of skill in the art for isolating and sequencingcDNA corresponding to human genes. An albumin protein of the inventionmay include the amino acid sequence of human serum albumin (HSA),provided in SEQ ID NO: 69 or SEQ ID NO: 70, or the amino acid sequenceof mouse serum albumin (MSA), provided in SEQ ID NO: 71, or a variant orfragment thereof, preferably a functional variant or fragment thereof. Afragment or variant may or may not be functional, or may retain thefunction of albumin to some degree. For example, a fragment or variantmay retain the ability to bind to an albumin receptor, such as HSA orMSA, by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or105% of the ability of the parent albumin (e.g., the parent albumin fromwhich the fragment or variant is derived). Relative binding ability maybe determined by methods known in the art, such as by surface plasmonresonance.

The albumin protein may be a naturally-occurring polymorphic variant ofan albumin protein, such as human serum albumin. Generally, variants orfragments of human serum albumin will have at least 5%, 10%, 15%, 20%,30%, 40%, 50%, 60%, or 70%, and preferably 80%, 90%, 95%, 100%, or 105%or more of human serum albumin or mouse serum albumin's ligand bindingactivity.

The albumin protein may include the amino acid sequence of bovine serumalbumin. Bovine serum albumin proteins include any albumin having anamino acid sequence naturally occurring in cows, for example, asdescribed by Swissprot accession number P02769, and variants thereof asdefined herein. Bovine serum albumin proteins also includes fragments offull-length bovine serum albumin or variants thereof, as defined herein.

The albumin protein may comprise the sequence of an albumin derived fromone of serum albumin from dog (e.g., Swissprot accession numberP49822-1), pig (e.g., Swissprot accession number P08835-1), goat (e.g.,Sigma product no. A2514 or A4164), cat (e.g., Swissprot accession numberP49064-1), chicken (e.g., Swissprot accession number P19121-1),ovalbumin (e.g., chicken ovalbumin) (e.g., Swissprot accession numberP01012-1), turkey ovalbumin (e.g., Swissprot accession number O73860-1),donkey (e.g., Swissprot accession number Q5XLE4-1), guinea pig (e.g.,Swissprot accession number Q6WDN9-1), hamster (e.g., as described inDeMarco et al. International Journal for Parasitology 37(11): 1201-1208(2007)), horse (e.g., Swissprot accession number P35747-1), rhesusmonkey (e.g., Swissprot accession number Q28522-1), mouse (e.g.,Swissprot accession number P07724-1), pigeon (e.g., as defined by Khanet al. Int. J. Biol. Macromol. 30(3-4), 171-8 (2002)), rabbit (e.g.,Swissprot accession number P49065-1), rat (e.g., Swissprot accessionnumber P02770-1) or sheep (e.g., Swissprot accession number P14639-1),and includes variants and fragments thereof as defined herein.

Many naturally-occurring mutant forms of albumin are known to thoseskilled in the art. Naturally-occurring mutant forms of albumin aredescribed in, for example, Peters, et al. All About Albumin:Biochemistry, Genetics and Medical Applications, Academic Press, Inc.,San Diego, Calif., p. 170-181 (1996).

Albumin proteins of the invention include variants ofnaturally-occurring albumin proteins. A variant albumin refers to analbumin protein having at least one amino acid mutation, such as anamino acid mutation generated by an insertion, deletion, orsubstitution, either conservative or non-conservative, provided thatsuch changes result in an albumin protein for which at least one basicproperty has not been significantly altered (e.g., has not been alteredby more than 5%, 10%, 15%, 20%, 25%, 30%, 35%, or 40%). Exemplaryproperties which may define the activity of an albumin protein includebinding activity (e.g., including binding specificity or affinity tobilirubin, or a fatty acid such as a long-chain fatty acid), osmolarity,or behavior in a certain pH-range.

Typically an albumin protein variant will have at least 40%, at least50%, at least 60%, and preferably at least 70%, at least 80%, at least90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least99% amino acid sequence identity with a naturally-occurring albuminprotein, such as the albumin protein of any one of SEQ ID NOs: 69-71.

Methods for the production and purification of recombinant humanalbumins are well-established (Sleep et al. Biotechnology, 8(1):42-6(1990)), and include the production of recombinant human albumin forpharmaceutical applications (Bosse et al. J Clin Pharmacol 45(1):57-67(2005)). The three-dimensional structure of HSA has been elucidated byX-ray crystallography (Carter et al. Science. 244(4909): 1195-8 (1998));Sugio et al. Protein Eng. 12(6):439-46 (1999)). The HSA polypeptidechain has 35 cysteine residues, which form 17 disulfide bonds, and oneunpaired (e.g., free) cysteine at position 34 of the mature protein.Cys-34 of HSA has been used for conjugation of molecules to albumin(Leger et al. Bioorg Med Chem Lett 14(17):4395-8 (2004); Thibaudeau etal. Bioconjug Chem 16(4):1000-8 (2005)), and provides a site forsite-specific conjugation.

(Human serum albumin (HSA), variant 1) SEQ ID NO: 69DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL(Human serum albumin (HSA), variant 2) SEQ ID NO: 70RGVFRRDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL (Mouse serum albumin (MSA))SEQ ID NO: 71 RGVFRREAHKSEIAHRYNDLGEQHFKGLVLIAFSQYLQKCSYDEHAKLVQEVTDFAKTCVADESAANCDKSLHTLFGDKLCAIPNLRENYGELADCCTKQEPERNECFLQHKDDNPSLPPFERPEAEAMCTSFKENPTTFMGHYLHEVARRHPYFYAPELLYYAEQYNEILTQCCAEADKESCLTPKLDGVKEKALVSSVRQRMKCSSMQKFGERAFKAWAVARLSQTFPNADFAEITKLATDLTKVNKECCHGDLLECADDRAELAKYMCENQATISSKLQTCCDKPLLKKAHCLSEVEHDTMPADLPAIAADFVEDQEVCKNYAEAKDVFLGTFLYEYSRRHPDYSVSLLLRLAKKYEATLEKCCAEANPPACYGTVLAEFQPLVEEPKNLVKTNCDLYEKLGEYGFQNAILVRYTQKAPQVSTPTLVEAARNLGRVGTKCCTLPEDQRLPCVEDYLSAILNRVCLLHEKTPVSEHVTKCCSGSLVERRPCFSALTVDETYVPKEFKAETFTFHSDICTLPEKEKQIKKQTALAELVKHKPKATAEQLKTVMDDFAQFLDTCCKAADKDTCFSTEGPNLVTRCKDALA

Conjugation of Albumin Proteins

An albumin protein of the invention may be conjugated to (e.g., byway ofa covalent bond) to any compound of the invention (e.g., byway of thelinker portion of a neuraminidase inhibitor monomer or dimer). Thealbumin protein may be conjugated to any compound of the invention byany method well-known to those of skill in the art for producingsmall-molecule-protein conjugates. This may include covalent conjugationto a solvent-exposed amino acid, such as a solvent exposed cysteine orlysine. For example, human serum albumin may be conjugated to a compoundof the invention by covalent linkage to the sulfur atom corresponding toCys34 of SEQ ID NO: 69 or Cys40 of SEQ ID NO: 70. An albumin protein ofthe invention may be conjugated to any compound of the invention by wayof an amino acid located within 10 amino acid residues of the C-terminalor N-terminal end of the albumin protein. An albumin protein may includea C-terminal or N-terminal polypeptide fusion of 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 15, or 20 or more amino acid. The C-terminal or N-terminalpolypeptide fusion may include one or more solvent-exposed cysteine orlysine residues, which may be used for covalent conjugation of acompound of the invention (e.g., conjugation to a neuraminidaseinhibitor monomer or dimer, including by way of a linker).

Albumin proteins of the invention include any albumin protein which hasbeen engineered to include one or more solvent-exposed cysteine orlysine residues, which may provide a site for conjugation to a compoundof the invention (e.g., conjugation to a neuraminidase inhibitor monomeror dimer, including by way of a linker). Most preferably, the albuminprotein will contain a single solvent-exposed cysteine or lysine, thusenabling site-specific conjugation of a compound of the invention.Exemplary methods for the production of engineered variants of albuminproteins that include one or more conjugation-competent cysteineresidues are provided in U.S. Patent Application No. 2017/0081389, whichis incorporated herein by reference in its entirety. Briefly, preferredalbumin protein variants are those comprising a single, solvent-exposed,unpaired (e.g., free) cysteine residue, thus enabling site-specificconjugation of a linker to the cysteine residue.

Albumin proteins which have been engineered to enable chemicalconjugation to a solvent-exposed, unpaired cysteine residue include thefollowing albumin protein variants:

(a) an albumin protein having a substitution of a non-cysteine aminoacid residue with a cysteine at an amino acid residue corresponding toany of L585, D1, A2, D562, A364, A504, E505, T79, E86, D129, D549, A581,D121, E82, S270, Q397, and A578 of SEQ ID NO: 69;

(b) an albumin protein having an insertion of a cysteine at a positionadjacent the N- or C-terminal side of an amino acid residuecorresponding to any of L585, D1, A2, D562, A364, A504, E505, T79, E86,D129, D549, A581, D121, E82, S270, Q397, and A578 of SEQ ID NO: 69;

(c) an albumin protein engineered to have an unpaired cysteine having afree thiol group at a residue corresponding to any of C369, C361, C91,C177, C567, C316, C75, C169, C124, or C558 of SEQ ID NO: 69, and whichmay or may not be generated by deletion or substitution of a residuecorresponding to C360, C316, C75, C168, C558, C361, C91, C124, C169, orC567 of SEQ ID NO: 69; and/or

(d) addition of a cysteine to the N- or C-terminus of an albuminprotein.

In some embodiments of the invention, the net result of thesubstitution, deletion, addition, or insertion events of (a), (b), (c)and/or (d) is that the number of conjugation competent cysteine residuesof the polypeptide sequence is increased relative to the parent albuminsequence. In some embodiments of the invention, the net result of thesubstitution, deletion, addition, or insertion events of (a), (b), (c)and/or (d) is that the number of conjugation competent-cysteine residuesof the polypeptide sequence is one, thus enabling site-specificconjugation.

Preferred albumin protein variants also include albumin proteins havinga single solvent-exposed lysine residue, thus enabling site-specificconjugation of a linker to the lysine residue. Such variants may begenerated by engineering an albumin protein, including any of themethods previously described (e.g., insertion, deletion, substitution,or C-terminal or N-terminal fusion).

Albumin Protein-Binding Peptides

Conjugation of a biologically-active compound to an albuminprotein-binding peptide can alter the pharmacodynamics of thebiologically-active compound, including the alteration of tissue uptake,penetration, and diffusion. In a preferred embodiment, conjugation of analbumin protein-binding peptide to a compound of the invention (e.g., aneuraminidase inhibitor monomer or dimer, byway of a linker) increasesthe efficacy or decreases the toxicity of the compound, as compared tothe compound alone.

Albumin protein-binding peptides of the invention include anypolypeptide having an amino acid sequence of 5 to 50 (e.g., 5 to 40, 5to 30, 5 to 20, 5 to 15, 5 to 10, 10 to 50, 10 to 30, or 10 to 20) aminoacid residues that has affinity for and functions to bind an albuminprotein, such as any of the albumin proteins described herein.Preferably, the albumin protein-binding peptide binds to a naturallyoccurring serum albumin, most preferably human serum albumin. An albuminprotein-binding peptide can be of different origins, e.g., synthetic,human, mouse, or rat. Albumin protein-binding peptides of the inventioninclude albumin protein-binding peptides which have been engineered toinclude one or more (e.g., two, three, four, or five) solvent-exposedcysteine or lysine residues, which may provide a site for conjugation toa compound of the invention (e.g., conjugation to a neuraminidaseinhibitor monomer or dimer, including byway of a linker). Mostpreferably, the albumin protein-binding peptide will contain a singlesolvent-exposed cysteine or lysine, thus enabling site-specificconjugation of a compound of the invention. Albumin protein-bindingpeptides may include only naturally occurring amino acid residues, ormay include one or more non-naturally occurring amino acid residues.Where included, a non-naturally occurring amino acid residue (e.g., theside chain of a non-naturally occurring amino acid residue) may be usedas the point of attachment for a compound of the invention (e.g., aneuraminidase inhibitor monomer or dimer, including by way of a linker).Albumin protein-binding peptides of the invention may be linear orcyclic. Albumin protein-binding peptides of the invention include anyalbumin protein-binding peptides known to one of skill in the art,examples of which, are provided herein.

Albumin protein-binding peptide, and conjugates including an albuminprotein-binding peptide, preferably bind an albumin protein (e.g., humanserum albumin) with an affinity characterized by a dissociationconstant, Kd, that is less than about 100 μM, preferably less than about100 nM, and most preferably do not substantially bind other plasmaproteins. Specific examples of such compounds are linear or cyclicpeptides, preferably between about 10 and 20 amino acid residues inlength, optionally modified at the N-terminus or C-terminus or both.

Albumin protein-binding peptides include linear and cyclic peptidescomprising the following general formulae, wherein Xaa is any aminoacid:

SEQ ID NO: 74 Xaa-Xaa-Cys-Xaa-Xaa-Xaa-Xaa-Xaa-Cys-Xaa-Xaa-Phe-Cys-Xaa-Asp-Trp-Pro-Xaa-Xaa-Xaa-Ser-Cys SEQ ID NO: 75Val-Cys-Tyr-Xaa-Xaa-Xaa-Ile-Cys-Phe SEQ ID NO: 76Cys-Tyr-Xaa-Pro-Gly-Xaa-Cys SEQ ID NO: 77Asp-Xaa-Cys-Leu-Pro-Xaa-Trp-Gly-Cys-Leu-Trp SEQ ID NO: 78Trp-Cys-Asp-Xaa-Xaa-Leu-Xaa-Ala-Xaa-Asp-Leu-Cys SEQ ID NO: 79Asp-Leu-Val-Xaa-Leu-Gly-Leu-Glu-Cys-TrpAlbumin protein-binding peptides of the invention further include any ofthe following peptide sequences, which may be linear or cyclic:

SEQ ID NO: 80 DLCLRDWGCLW SEQ ID NO: 81 DICLPRWGCLW SEQ ID NO: 82MEDICLPRWGCLWGD SEQ ID NO: 83 QRLMEDICLPRWGCLWEDDE SEQ ID NO: 84QGLIGDICLPRWGCLWGRSV SEQ ID NO: 85 QGLIGDICLPRWGCLWGRSVK SEQ ID NO: 86EDICLPRWGCLWEDD SEQ ID NO: 87 RLMEDICLPRWGCLWEDD SEQ ID NO: 88MEDICLPRWGCLWEDD SEQ ID NO: 89 MEDICLPRWGCLWED SEQ ID NO: 90RLMEDICLARWGCLWEDD SEQ ID NO: 91 EVRSFCTRWPAEKSCKPLRG SEQ ID NO: 92RAPESFVCYWETICFERSEQ SEQ ID NO: 93 EMCYFPGICWM

Albumin protein-binding peptides of SEQ ID NOs: 74-93 may furtherinclude additional amino acids at the N-terminus (Xaa)x and/oradditional amino acids at the C-terminus (Xaa)z, wherein Xaa is anyamino acid and x and z are a whole number greater or equal to zero,generally less than 100, preferably less than 10 and more preferably 0,1, 2, 3, 4 or 5.

Further exemplary albumin protein-binding peptides are provided in U.S.Patent Application No. 2005/0287153, which is incorporated herein byreference in its entirety.

Conjugation of Albumin Protein-Binding Peptides

An albumin protein-binding peptide of the invention may be conjugated to(e.g., by way of a covalent bond) to any compound of the invention(e.g., by way of the linker portion of a neuraminidase inhibitor monomeror dimer). The albumin protein-binding peptide may be conjugated to anycompound of the invention by any method known to those of skill in theart for producing peptide-small molecule conjugates. This may includecovalent conjugation to the side chain group of an amino acid residue,such as a cysteine, a lysine, or a non-natural amino acid. Alternately,covalent conjugation may occur at the C-terminus (e.g., to theC-terminal carboxylic acid, or to the side chain group of the C-terminalresidue) or at the N-terminus (e.g., to the N-terminal amino group, orto the side chain group of the N-terminal amino acid).

V. Linkers

A linker refers to a linkage or connection between two or morecomponents in a conjugate described herein (e.g., between twoneuraminidase inhibitors in a conjugate described herein, between aneuraminidase inhibitor and an Fc domain or an albumin protein in aconjugate described herein, and between a dimer of two neuraminidaseinhibitors and an Fc domain or an albumin protein in a conjugatedescribed herein).

Linkers in Conjugates Having an Fc Domain or an Albumin ProteinCovalently Linked to Dimers of Neuraminidase Inhibitors

In a conjugate containing an Fc domain monomer, an Fc domain, anFc-binding peptide, an albumin protein, or an albumin protein-bindingpeptide covalently linked to one or more dimers of neuraminidaseinhibitors as described herein, a linker in the conjugate (e.g., L orL′) may be a branched structure. As described further herein, a linkerin a conjugate described herein (e.g., L or L′) may be a multivalentstructure, e.g., a divalent or trivalent structure having two or threearms, respectively. In some embodiments when the linker has three arms,two of the arms may be attached to the first and second neuraminidaseinhibitors and the third arm may be attached to the Fc domain monomer,and Fc domain, an Fc-binding peptide, an albumin protein, or an albuminprotein-binding peptide. In some embodiments when the linker has twoarms, one arm may be attached to an Fc domain or an albumin protein andthe other arm may be attached to one of the two neuraminidaseinhibitors. In other embodiments, a linker with two arms may be used toattach the two neuraminidase inhibitors on a conjugate containing an Fcdomain or albumin protein covalently linked to one or more dimers ofneuraminidase inhibitors.

In some embodiments, a linker in a conjugate having an Fc domain or analbumin protein covalently linked to one or more dimers of neuraminidaseinhibitors is described by formula (D-L-I):

wherein L^(A) is described by formulaG^(A1)-(Z^(A1))_(g1)-(Y^(A1))_(h1)-(Z^(A2))_(i1)-(Y^(A2))_(j1)-(Z^(A3))_(k1)-(Y^(A3))_(l1)-(Z^(A4))_(m1)-(Y^(A4))_(n1)-(Z^(A5))_(o1)-G^(A2);L^(B) is described by formulaG^(B1)-(Z^(B1))_(g2)-(Y^(B1))_(h2)-(Z^(B2))_(i2)-(Y^(B2))_(j2)-(Z^(B3))_(k2)-(Y^(B3))_(l2)-(Z^(B4))_(m2)-(Y^(B4))_(n2)-(Z^(B5))_(o2)-G^(B2);L^(C) is described by formulaG^(c1)-(Z^(c1))_(g3)-(Y^(c1))_(h3)-(Z^(C2))_(i3)-(Y^(C2))_(j3)-(Z^(C3))_(k3)-(Y^(C3))_(l3)-(Z^(C4))_(m3)-(Y^(C4))_(n3)-(Z^(C5))_(o3)-G^(C2);G^(A1) is a bond attached to Q in formula (D-L-I); G^(A2) is a bondattached to the first neuraminidase inhibitor (e.g., A₁); G^(B1) is abond attached to Q in formula (D-L-I); G^(B2) is a bond attached to thesecond neuraminidase inhibitor (e.g., A₂); G^(C1) is a bond attached toQ in formula (D-L-I); G^(C2) is a bond attached to an Fc domain monomer,an Fc domain, an Fc-binding peptide, an albumin protein, or an albuminprotein-binding peptide, or a functional group capable of reacting witha functional group conjugated to an Fc domain monomer, an Fc domain, anFc-binding peptide, an albumin protein, or an albumin protein-bindingpeptide (e.g., maleimide and cysteine, amine and activated carboxylicacid, thiol and maleimide, activated sulfonic acid and amine, isocyanateand amine, azide and alkyne, and alkene and tetrazine); each of Z^(A1),Z^(A2), Z^(A3), Z^(A4), Z^(A5), Z^(B1), Z^(B2), Z^(B3), Z^(B4), Z^(B5),Z^(C1), Z^(C2), Z^(C3), Z^(C4) and Z^(C5) is, independently, optionallysubstituted C1-C20 alkylene, optionally substituted C1-C20heteroalkylene, optionally substituted C2-C20 alkenylene, optionallysubstituted C2-C20 heteroalkenylene, optionally substituted C2-C20alkynylene, optionally substituted C2-C20 heteroalkynylene, optionallysubstituted C3-C20 cycloalkylene, optionally substituted C3-C20heterocycloalkylene, optionally substituted C4-C20 cycloalkenylene,optionally substituted C4-C20 heterocycloalkenylene, optionallysubstituted C8-C20 cycloalkynylene, optionally substituted C8-C20heterocycloalkynylene, optionally substituted C5-C15 arylene, oroptionally substituted C2-C15 heteroarylene; each of Y^(A1), Y^(A2),Y^(A3), Y^(A4), Y^(B1), Y^(B2), Y^(B3), Y^(B4), Y^(C1), Y^(C2), Y^(C3),and Y^(C4) is, independently, O, S, NR^(i), P, carbonyl, thiocarbonyl,sulfonyl, phosphate, phosphoryl, or imino; R^(i) is H, optionallysubstituted C1-C20 alkyl, optionally substituted C1-C20 heteroalkyl,optionally substituted C2-C20 alkenyl, optionally substituted C2-C20heteroalkenyl, optionally substituted C2-C20 alkynyl, optionallysubstituted C2-C20 heteroalkynyl, optionally substituted C3-C20cycloalkyl, optionally substituted C3-C20 heterocycloalkyl, optionallysubstituted C4-C20 cycloalkenyl, optionally substituted C4-C20heterocycloalkenyl, optionally substituted C8-C20 cycloalkynyl,optionally substituted C8-C20 heterocycloalkynyl, optionally substitutedC5-C15 aryl, or optionally substituted C2-C15 heteroaryl; each of g1,h1, i1, j1, k1, l1, m1, n1, o1, g2, h2, i2, j2, k2, l2, m2, n2, o2, g3,h3, i3, j3, k3, l3, m3, n3, and o3 is, independently, 0 or 1; Q is anitrogen atom, optionally substituted C1-C20 alkylene, optionallysubstituted C1-C20 heteroalkylene, optionally substituted C2-C20alkenylene, optionally substituted C2-C20 heteroalkenylene, optionallysubstituted C2-C20 alkynylene, optionally substituted C2-C20heteroalkynylene, optionally substituted C3-C20 cycloalkylene,optionally substituted C3-C20 heterocycloalkylene, optionallysubstituted C4-C20 cycloalkenylene, optionally substituted C4-C20heterocycloalkenylene, optionally substituted C8-C20 cycloalkynylene,optionally substituted C8-C20 heterocycloalkynylene, optionallysubstituted C5-C15 arylene, or optionally substituted C2-C15heteroarylene.

In some embodiments, L^(C) may have two points of attachment to the Fcdomain (e.g., two G^(C2)). In some embodiments, L includes apolyethylene glycol (PEG) linker. A PEG linker includes a linker havingthe repeating unit structure (—CH₂CH₂O—)_(n), where n is an integer from2 to 100. A polyethylene glycol linker may covalently join aneuraminidase inhibitor and E (e.g., in a conjugate of any one offormulas (M-I)-(M-X)). A polyethlylene glycol linker may covalently joina first neuraminidase inhibitor and a second neuraminidase inhibitor(e.g., in a conjugate of any one of formulas (D-I)-(D-X)). Apolyethylene glycol linker may covalently join a neuraminidase inhibitordimer and E (e.g., in a conjugate of any one of formulas (D-I)-(D-X)). Apolyethylene glycol linker may selected any one of PEG₂ to PEG₁₀₀ (e.g.,PEG₂, PEG₃, PEG₄, PEG₅, PEG₅-PEG₁₀, PEG₁₀-PEG₂₀, PEG₂₀-PEG₃₀,PEG₃₀-PEG₄₀, PEG₅₀-PEG₆₀, PEG₆₀-PEG₇₀, PEG₇₀-PEG₈₀, PEG₈₀-PEG₉₀,PEG₉₀-PEG₁₀₀). In some embodiments, L^(C) includes a PEG linker, whereL^(C) is covalently attached to each of Q and E.

Linkers of formula (D-L-I) that may be used in conjugates describedherein include, but are not limited to

wherein z₁ and z₂ are each, independently, and integer from 1 to 20; andR₉ is selected from H, C1-C20 alkyl, C3-C20 cycloalkyl, C3-C20heterocycloalkyl; C5-C15 aryl, and C2-C15 heteroaryl.

Linkers of the formula (D-L-I) may also include any of

Linkers in Conjugates Having an Fc Domain or an Albumin ProteinCovalently Linked to Monomers of Neuraminidase Inhibitors

In a conjugate containing an Fc domain monomer, an Fc domain, anFc-binding peptide, an albumin protein, or an albumin protein-bindingpeptide covalently linked to one or more monomers of neuraminidaseinhibitors as described herein, a linker in the conjugate (e.g., L, orL′) may be a divalent structure having two arms. One arm in a divalentlinker may be attached to the monomer of neuraminidase inhibitor and theother arm may be attached to the Fc domain monomer, and Fc domain, anFc-binding peptide, an albumin protein, or an albumin protein-bindingpeptide. In some embodiments, the one or more monomers of neuraminidaseinhibitors in the conjugates described herein may each be,independently, connected to an atom in the Fc domain monomer, and Fcdomain, an Fc-binding peptide, an albumin protein, or an albuminprotein-binding peptide.

In some embodiments, a linker is described by formula (M-L-I):

J¹-(Q¹)_(g)-(T¹)_(h)-(Q²)_(i)-(T²)_(j)-(Q³)_(k)-(T³)_(l)-(Q⁴)_(m)-(T⁴)_(n)-(Q⁵)_(o)-J²

wherein J¹ is a bond attached to a neuraminidase inhibitor; J² is a bondattached to an Fc domain monomer, an Fc domain, an Fc-binding peptide,an albumin protein, or an albumin protein-binding peptide, or afunctional group capable of reacting with a functional group conjugatedto an Fc domain monomer, an Fc domain, an Fc-binding peptide, an albuminprotein, or an albumin protein-binding peptide (e.g., maleimide andcysteine, amine and activated carboxylic acid, thiol and maleimide,activated sulfonic acid and amine, isocyanate and amine, azide andalkyne, and alkene and tetrazine); each of Q¹, Q², Q³, Q⁴, and Q⁵ is,independently, optionally substituted C1-C20 alkylene, optionallysubstituted C1-C20 heteroalkylene, optionally substituted C2-C20alkenylene, optionally substituted C2-C20 heteroalkenylene, optionallysubstituted C2-C20 alkynylene, optionally substituted C2-C20heteroalkynylene, optionally substituted C3-C20 cycloalkylene,optionally substituted C3-C20 heterocycloalkylene, optionallysubstituted C4-C20 cycloalkenylene, optionally substituted C4-C20heterocycloalkenylene, optionally substituted C8-C20 cycloalkynylene,optionally substituted C8-C20 heterocycloalkynylene, optionallysubstituted C5-C15 arylene, or optionally substituted C2-C15heteroarylene; each of T¹, T², T³, T⁴ is, independently, O, S, NR^(i),P, carbonyl, thiocarbonyl, sulfonyl, phosphate, phosphoryl, or imino;R^(i) is H, optionally substituted C1-C20 alkyl, optionally substitutedC1-C20 heteroalkyl, optionally substituted C2-C20 alkenyl, optionallysubstituted C2-C20 heteroalkenyl, optionally substituted C2-C20 alkynyl,optionally substituted C2-C20 heteroalkynyl, optionally substitutedC3-C20 cycloalkyl, optionally substituted C3-C20 heterocycloalkyl,optionally substituted C4-C20 cycloalkenyl, optionally substitutedC4-C20 heterocycloalkenyl, optionally substituted C8-C20 cycloalkynyl,optionally substituted C8-C20 heterocycloalkynyl, optionally substitutedC5-C15 aryl, or optionally substituted C2-C15 heteroaryl; and each of g,h, i, j, k, l, m, n, and o is, independently, 0 or 1.

In some embodiments, J² may have two points of attachment to the Fcdomain monomer, and Fc domain, an Fc-binding peptide, an albuminprotein, or an albumin protein-binding peptide (e.g., two J²).

Linkers of formula (M-L-l) that may be used in conjugates describedherein include, but are not limited to,

wherein d is an integer from 1 to 20 (e.g., d is 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20).

Linkers of formula (M-L-l) that may be used in conjugates describedherein include, but are not limited to,

wherein each of d and e is, independently, an integer from 1 to 26.

Linking Groups

In some embodiments, a linker provides space, rigidity, and/orflexibility between the neuraminidase inhibitors and the Fc domainmonomer, and Fc domain, an Fc-binding peptide, an albumin protein, or analbumin protein-binding peptide in the conjugates described here orbetween two neuraminidase inhibitors in the conjugates described herein.In some embodiments, a linker may be a bond, e.g., a covalent bond,e.g., an amide bond, a disulfide bond, a C—O bond, a C—N bond, a N—Nbond, a C—S bond, or any kind of bond created from a chemical reaction,e.g., chemical conjugation. In some embodiments, a linker (L or L′ asshown in any one of formulas (1)-(5), (D-I)-(D-X), (D′-I), (M-I)-(M-X),or (M′-I)) includes no more than 250 atoms (e.g., 1-2, 1-4, 1-6, 1-8,1-10, 1-12, 1-14, 1-16, 1-18, 1-20, 1-25, 1-30, 1-35, 1-40, 1-45, 1-50,1-55, 1-60, 1-65, 1-70, 1-75, 1-80, 1-85, 1-90, 1-95, 1-100, 1-110,1-120, 1-130, 1-140, 1-150, 1-160, 1-170, 1-180, 1-190, 1-200, 1-210,1-220, 1-230, 1-240, or 1-250 atom(s); 250, 240, 230, 220, 210, 200,190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 95, 90, 85, 80, 75,70, 65, 60, 55, 50, 45, 40, 35, 30, 28, 26, 24, 22, 20, 18, 16, 14, 12,10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 atom(s)). In some embodiments, a linker(L or L) includes no more than 250 non-hydrogen atoms (e.g., 1-2, 1-4,1-6, 1-8, 1-10, 1-12, 1-14, 1-16, 1-18, 1-20, 1-25, 1-30, 1-35, 1-40,1-45, 1-50, 1-55, 1-60, 1-65, 1-70, 1-75, 1-80, 1-85, 1-90, 1-95, 1-100,1-110, 1-120, 1-130, 1-140, 1-150, 1-160, 1-170, 1-180, 1-190, 1-200,1-210, 1-220, 1-230, 1-240, or 1-250 non-hydrogen atom(s); 250, 240,230, 220, 210, 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100,95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 28, 26, 24, 22,20, 18, 16, 14, 12, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 non-hydrogenatom(s)). In some embodiments, the backbone of a linker (L or L)includes no more than 250 atoms (e.g., 1-2, 1-4, 1-6, 1-8, 1-10, 1-12,1-14, 1-16, 1-18, 1-20, 1-25, 1-30, 1-35, 1-40, 1-45, 1-50, 1-55, 1-60,1-65, 1-70, 1-75, 1-80, 1-85, 1-90, 1-95, 1-100, 1-110, 1-120, 1-130,1-140, 1-150, 1-160, 1-170, 1-180, 1-190, 1-200, 1-210, 1-220, 1-230,1-240, or 1-250 atom(s); 250, 240, 230, 220, 210, 200, 190, 180, 170,160, 150, 140, 130, 120, 110, 100, 95, 90, 85, 80, 75, 70, 65, 60, 55,50, 45, 40, 35, 30, 28, 26, 24, 22, 20, 18, 16, 14, 12, 10, 9, 8, 7, 6,5, 4, 3, 2, or 1 atom(s)). The “backbone” of a linker refers to theatoms in the linker that together form the shortest path from one partof the conjugate to another part of the conjugate. The atoms in thebackbone of the linker are directly involved in linking one part of theconjugate to another part of the conjugate. For examples, hydrogen atomsattached to carbons in the backbone of the linker are not considered asdirectly involved in linking one part of the conjugate to another partof the conjugate.

Molecules that may be used to make linkers (L or L′) include at leasttwo functional groups, e.g., two carboxylic acid groups. In someembodiments of a trivalent linker, two arms of a linker may contain twodicarboxylic acids, in which the first carboxylic acid may form acovalent linkage with the first neuraminidase inhibitor in the conjugateand the second carboxylic acid may form a covalent linkage with thesecond neuraminidase inhibitor in the conjugate, and the third arm ofthe linker may for a covalent linkage (e.g., a C—O bond) with an Fcdomain monomer, an Fc domain, an Fc-binding peptide, an albumin protein,or an albumin protein-binding peptide in the conjugate. In someembodiments of a divalent linker, the divalent linker may contain twocarboxylic acids, in which the first carboxylic acid may form a covalentlinkage with one component (e.g., a neuraminidase inhibitor) in theconjugate and the second carboxylic acid may form a covalent linkage(e.g., a C—S bond or a C—N bond) with another component (e.g., an Fcdomain monomer, an Fc domain, an Fc-binding peptide, an albumin protein,or an albumin protein-binding peptide) in the conjugate.

In some embodiments, dicarboxylic acid molecules may be used as linkers(e.g., a dicarboxylic acid linker). For example, in a conjugatecontaining an Fc domain monomer, an Fc domain, an Fc-binding peptide, analbumin protein, or an albumin protein-binding peptide covalently linkedto one or more dimers of neuraminidase inhibitors, the first carboxylicacid in a dicarboxylic acid molecule may form a covalent linkage with ahydroxyl or amine group of the first neuraminidase inhibitor and thesecond carboxylic acid may form a covalent linkage with a hydroxyl oramine group of the second neuraminidase inhibitor.

Examples of dicarboxylic acids molecules that may be used to formlinkers include, but are not limited to,

wherein n is an integer from 1 to 20 (e.g., n is 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20).

Other examples of dicarboxylic acids molecules that may be used to formlinkers include, but are not limited to,

In some embodiments, dicarboxylic acid molecules, such as the onesdescribed herein, may be further functionalized to contain one or moreadditional functional groups. Dicarboxylic acids may be furtherfunctionalized, for example, to provide an attachment point to an Fcdomain monomer, an Fc domain, an Fc-binding peptide, an albumin protein,or an albumin protein-binding peptide (e.g., byway of a linker, such asa PEG linker).

In some embodiments, when the neuraminidase inhibitor is attached to Fcdomain monomer, an Fc domain, an Fc-binding peptide, an albumin protein,or an albumin protein-binding peptide, the linking group may comprise amoiety comprising a carboxylic acid moiety and an amino moiety that arespaced by from 1 to 25 atoms. Examples of such linking groups include,but are not limited to,

wherein n is an integer from 1 to 20 (e.g., n is 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20).

In some embodiments, a linking group may including a moiety including acarboxylic acid moiety and an amino moiety, such as the ones describedherein, may be further functionalized to contain one or more additionalfunctional groups. Such linking groups may be further functionalized,for example, to provide an attachment point to an Fc domain monomer, anFc domain, an Fc-binding peptide, an albumin protein, or an albuminprotein-binding peptide (e.g., by way of a linker, such as a PEGlinker). In some embodiments, when the neuraminidase inhibitor isattached to Fc domain monomer, an Fc domain, an Fc-binding peptide, analbumin protein, or an albumin protein-binding peptide, the linkinggroup may comprise a moiety comprising two or amino moieties (e.g., adiamino moiety) that are spaced by from 1 to 25 atoms. Examples of suchlinking groups include, but are not limited to,

wherein n is an integer from 1 to 20 (e.g., n is 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20).

In some embodiments, a linking group may including a diamino moiety,such as the ones described herein, may be further functionalized tocontain one or more additional functional groups. Such diamino linkinggroups may be further functionalized, for example, to provide anattachment point to an Fc domain monomer, an Fc domain, an Fc-bindingpeptide, an albumin protein, or an albumin protein-binding peptide(e.g., byway of a linker, such as a PEG linker).

In some embodiments, a molecule containing an azide group may be used toform a linker, in which the azide group may undergo cycloaddition withan alkyne to form a 1,2,3-triazole linkage. In some embodiments, amolecule containing an alkyne group may be used to form a linker, inwhich the alkyne group may undergo cycloaddition with an azide to form a1,2,3-triazole linkage. In some embodiments, a molecule containing amaleimide group may be used to form a linker, in which the maleimidegroup may react with a cysteine to form a C—S linkage. In someembodiments, a molecule containing one or more sulfonic acid groups maybe used to form a linker, in which the sulfonic acid group may form asulfonamide linkage with the linking nitrogen in a neuraminidaseinhibitor. In some embodiments, a molecule containing one or moreisocyanate groups may be used to form a linker, in which the isocyanategroup may form a urea linkage with the linking nitrogen in aneuraminidase inhibitor. In some embodiments, a molecule containing oneor more haloalkyl groups may be used to form a linker, in which thehaloalkyl group may form a covalent linkage, e.g., C—N and C—O linkages,with a neuraminidase inhibitor.

In some embodiments, a linker (L or L′) may comprise a synthetic groupderived from, e.g., a synthetic polymer (e.g., a polyethylene glycol(PEG) polymer). In some embodiments, a linker may comprise one or moreamino acid residues. In some embodiments, a linker may be an amino acidsequence (e.g., a 1-25 amino acid, 1-10 amino acid, 1-9 amino acid, 1-8amino acid, 1-7 amino acid, 1-6 amino acid, 1-5 amino acid, 1-4 aminoacid, 1-3 amino acid, 1-2 amino acid, or 1 amino acid sequence). In someembodiments, a linker (L or L′) may include one or more optionallysubstituted C1-C20 alkylene, optionally substituted C1-C20heteroalkylene (e.g., a PEG unit), optionally substituted C2-C20alkenylene (e.g., C2 alkenylene), optionally substituted C2-C20heteroalkenylene, optionally substituted C2-C20 alkynylene, optionallysubstituted C2-C20 heteroalkynylene, optionally substituted C3-C20cycloalkylene (e.g., cyclopropylene, cyclobutylene), optionallysubstituted C3-C20 heterocycloalkylene, optionally substituted C4-C20cycloalkenylene, optionally substituted C4-C20 heterocycloalkenylene,optionally substituted C8-C20 cycloalkynylene, optionally substitutedC8-C20 heterocycloalkynylene, optionally substituted C5-C15 arylene(e.g., C6 arylene), optionally substituted C2-C15 heteroarylene (e.g.,imidazole, pyridine), O, S, NR^(i) (R^(i) is H, optionally substitutedC1-C20 alkyl, optionally substituted C1-C20 heteroalkyl, optionallysubstituted C2-C20 alkenyl, optionally substituted C2-C20 heteroalkenyl,optionally substituted C2-C20 alkynyl, optionally substituted C2-C20heteroalkynyl, optionally substituted C3-C20 cycloalkyl, optionallysubstituted C3-C20 heterocycloalkyl, optionally substituted C4-C20cycloalkenyl, optionally substituted C4-C20 heterocycloalkenyl,optionally substituted C8-C20 cycloalkynyl, optionally substitutedC8-C20 heterocycloalkynyl, optionally substituted C5-C15 aryl, oroptionally substituted C2-C15 heteroaryl), P, carbonyl, thiocarbonyl,sulfonyl, phosphate, phosphoryl, or imino.

Conjugation Chemistries

Neuraminidase inhibitor monomer or dimers (e.g., in a conjugate of anyone of formulas (1)-(5), (D-I)-(D-X), (D′-I), (M-I)-(M-X), or (M′-I))may be conjugated to an Fc domain monomer, an Fc domain, an Fc-bindingpeptide, an albumin protein, or an albumin protein-binding peptide,e.g., byway of a linker, by any standard conjugation chemistries knownto those of skill in the art. The following conjugation chemistries arespecifically contemplated, e.g., for conjugation of a PEG linker (e.g.,a functionalized PEG linker) to an Fc domain monomer, an Fc domain, anFc-binding peptide, an albumin protein, or an albumin protein-bindingpeptide.

Covalent conjugation of two or more components in a conjugate using alinker may be accomplished using well-known organic chemical synthesistechniques and methods. Complementary functional groups on twocomponents may react with each other to form a covalent bond. Examplesof complementary reactive functional groups include, but are not limitedto, e.g., maleimide and cysteine, amine and activated carboxylic acid,thiol and maleimide, activated sulfonic acid and amine, isocyanate andamine, azide and alkyne, and alkene and tetrazine. Site-specificconjugation to a polypeptide (e.g., an Fc domain monomer, an Fc domain,an Fc-binding peptide, an albumin protein, or an albumin protein-bindingpeptide) may be accomplished using techniques known in the art.Exemplary techniques for site-specific conjugation of a small moleculeto an Fc domain are provided in Agarwall. P., et al. Bioconjugate Chem.26:176-192 (2015).

Other examples of functional groups capable of reacting with aminogroups include, e.g., alkylating and acylating agents. Representativealkylating agents include: (i) an α-haloacetyl group, e.g., XCH₂CO—(where X=Br, Cl, or I); (ii) a N-maleimide group, which may react withamino groups either through a Michael type reaction or through acylationby addition to the ring carbonyl group; (iii) an aryl halide, e.g., anitrohaloaromatic group; (iv) an alkyl halide; (v) an aldehyde or ketonecapable of Schiff's base formation with amino groups; (vi) an epoxide,e.g., an epichlorohydrin and a bisoxirane, which may react with amino,sulfhydryl, or phenolic hydroxyl groups; (vii) a chlorine-containing ofs-triazine, which is reactive towards nucleophiles such as amino,sulfhydryl, and hydroxyl groups; (viii) an aziridine, which is reactivetowards nucleophiles such as amino groups by ring opening; (ix) asquaric acid diethyl ester; and (x) an α-haloalkyl ether.

Examples of amino-reactive acylating groups include, e.g., (i) anisocyanate and an isothiocyanate; (ii) a sulfonyl chloride; (iii) anacid halide; (iv) an active ester, e.g., a nitrophenylester orN-hydroxysuccinimidyl ester, or derivatives thereof (e.g.,azido-PEG₂-PEG₄₀-NHS ester); (v) an acid anhydride, e.g., a mixed,symmetrical, or N-carboxyanhydride; (vi) an acylazide; and (vii) animidoester. Aldehydes and ketones may be reacted with amines to formSchiff's bases, which may be stabilized through reductive amination.

It will be appreciated that certain functional groups may be convertedto other functional groups prior to reaction, for example, to conferadditional reactivity or selectivity. Examples of methods useful forthis purpose include conversion of amines to carboxyls using reagentssuch as dicarboxylic anhydrides; conversion of amines to thiols usingreagents such as N-acetylhomocysteine thiolactone,S-acetylmercaptosuccinic anhydride, 2-iminothiolane, or thiol-containingsuccinimidyl derivatives; conversion of thiols to carboxyls usingreagents such as a -haloacetates; conversion of thiols to amines usingreagents such as ethylenimine or 2-bromoethylamine; conversion ofcarboxyls to amines using reagents such as carbodiimides followed bydiamines; and conversion of alcohols to thiols using reagents such astosyl chloride followed by transesterification with thioacetate andhydrolysis to the thiol with sodium acetate.

In some embodiments, a linker of the invention (e.g., L or L′, such asL^(C) of D-L-I), is conjugated (e.g., by any of the methods describedherein) to E (e.g., an Fc domain or albumin protein). In preferredembodiments of the invention, the linker is conjugated by way of: (a) athiourea linkage (i.e., —NH(C═S)NH—) to a lysine of E; (b) a carbamatelinkage (i.e., —NH(C═O)—O) to a lysine of E; (c) an amine linkage byreductive amination (i.e., —NHCH₂) between a lysine and E; (d) an amide(i.e., —NH—(C═O)CH₂) to a lysine of E; (e) a cysteine-maleimideconjugate between a maleimide of the linker to a cysteine of E; (f) anamine linkage by reductive amination (i.e., —NHCH₂) between the linkerand a carbohydrate of E (e.g., a glycosyl group of an Fc domain monomeror an Fc domain); (g) a rebridged cysteine conjugate, wherein the linkeris conjugated to two cysteines of E; (h) an oxime linkage between thelinker and a carbohydrate of E (e.g., a glycosyl group of an Fc domainmonomer or an Fc domain); (i) an oxime linkage between the linker and anamino acid residue of E; (j) an azido linkage between the linker and E;(k) direct acylation of a linker to E; or (I) a thioether linkagebetween the linker and E.

In some embodiments, a linker is conjugated to E, wherein the linkageincludes the structure —NH(C═NH)X—, wherein X is O, HN, or a bond. Insome embodiments, a linker is conjugated to E, wherein the linkagebetween the remainder of the linker and E includes the structure—NH(C═O)NH—.

In some embodiments, a linker (e.g., an active ester, e.g., anitrophenylester or N-hydroxysuccinimidyl ester, or derivatives thereof(e.g., a functionalized PEG linker (e.g., azido-PEG₂-PEG₄₀-NHS ester),is conjugated to E, with a T of (e.g., DAR) of between 0.5 and 10.0,e.g., 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7,1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2,3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7,4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1,6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5,7.6, 7.7, 7.8.0, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9,9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or 10.0. In theseinstances, the E-(PEG₂-PEG₄₀)-azide can react with an Int having aterminal alkyne linker (e.g., L, or L′, such as L^(C) of D-L-I) throughclick conjugation. During click conjugation, the copper-catalyzedreaction of the an azide (e.g., the Fc-(PEG₂-PEG₄₀)-azide) with thealkyne (e.g., the Int having a terminal alkyne linker (e.g., L or L′,such as L^(C) of D-L-I) forming a 5-membered heteroatom ring. In someembodiments, the linker conjugated to E is a terminal alkyne and isconjugated to an Int having a terminal azide. Exemplary preparations ofpreparations of E-(PEG₂-PEG₄₀)-azide are described in Examples 7, 8, 61,84, 88, and 124. Exemplary figures of conjugates prepared through clickconjugation are depicted in FIGS. 43 and 61. One of skill in the artwould readily understand the final product from a click chemistryconjugation.

Exemplary linking strategies (e.g., methods for linking a monomer or adimer of a neuraminidase inhibitor to E, such as, by way of a linker)are further depicted in FIGS. 1, 28, 29, 30, 43, and 61.

VI. Combination Therapies Antiviral Agents

In some embodiments, one or more antiviral agents may be administered incombination (e.g., administered substantially simultaneously (e.g., inthe same pharmaceutical composition or in separate pharmaceuticalcompositions) or administered separately at different times) with aconjugate described herein (e.g., a conjugate of any one of formulas(1)-(5), (D-I)-(D-X), (D′-I), (M-I)-(M-X), or (M′-I)).

In some embodiments, the antiviral agent is an antiviral agent for thetreatment of influenza virus. For example, the antiviral agent may be anM2 ion channel blocker, a neuraminidase inhibitor (e.g., a long-actingneuraminidase inhibitor), a polymerase inhibitor, a hemagglutinininhibitor, a fusion protein inhibitor, a COX-2 inhibitor, or a PPARagonist. The antiviral agent may target either the virus or the hostsubject. The antiviral agent for the treatment of influenza virus usedin combination with a conjugate described herein (e.g., a conjugate ofany one of formulas (1)-(5), (D-I)-(D-X), (D′-I), (M-I)-(M-X), or(M′-I)) may be selected from oseltamivir, zanamivir, peramivir,laninamivir, CS-8958, amantadine, rimantadine, cyanovirin-N, acap-dependent endonuclease inhibitor (e.g., baloxavir marboxil), apolymerase inhibitor (e.g., T-705), a PB2 inhibitor (e.g.,JNJ-63623872), a conjugated sialidase (e.g., DAS181), a thiazolide(e.g., nitazoxanide), a COX inhibitor, a PPAR agonist, ahemagglutinin-targeting antibody (e.g., a monoclonal antibody such asCR6261, CR8020, MEDI8852, MHAA4549A, or VIS410), or an siRNA targeting ahost or viral gene, or prodrugs thereof, or pharmaceutically acceptablesalts thereof.

Antiviral Vaccines

In some embodiments, any one of conjugates described herein (e.g., aconjugate of any one of formulas (1)-(5), (D-I)-(D-X), (D′-I),(M-I)-(M-X), or (M′-I)) is administered in combination with an antiviralvaccine (e.g., a composition that elicits an immune response in asubject directed against a virus). The antiviral vaccine may beadministered substantially simultaneously (e.g., in the samepharmaceutical composition or in separate pharmaceutical compositions)as the conjugates, or may be administered prior to or following theconjugates (e.g., within a period of 1 day, 2, days, 5, days, 1 week, 2weeks, 3 weeks, 1 month, 2 months, 6 months, or 12 months, or more).

In some embodiments the viral vaccine comprises an immunogen thatelicits an immune response in the subject against influenza virus A, B,C, or parainfluenza virus. In some embodiments the immunogen is aninactivated virus (e.g., the vaccine is a trivalent influenza vaccinethat contains purified and inactivated material influenza virus A, B, C,or parainfluenza virus or any combination thereof). In some embodimentsthe vaccine is given as an intramuscular injection. In some embodiments,the vaccine is a live virus vaccine that contains live viruses that havebeen attenuated (weakened). In some embodiments the vaccine isadministered as a nasal spray.

VII. Methods

Methods described herein include, e.g., methods of protecting against ortreating a viral infection (e.g., an influenza viral infection) in asubject and methods of preventing, stabilizing, or inhibiting the growthof viral particles. A method of treating a viral infection (e.g., aninfluenza viral infection) in a subject includes administering to thesubject a conjugate described herein (e.g., a conjugate of any one offormulas (1)-(5), (D-I)-(D-X), (D′-I), (M-I)-(M-X), or (M′-I)) or apharmaceutical composition thereof. In some embodiments, the viralinfection is cause by the influenza virus (e.g., influenza virus A, B,C, or parainfluenza virus). In some embodiments, the viral infection iscaused by a resistant strain of virus. A method of preventing,stabilizing, or inhibiting the growth of viral particles or preventingthe replication and spread of the virus includes contacting the virus ora site susceptible to viral growth with a conjugate described herein(e.g., a conjugate of any one of formulas (1)-(5), (D-I)-(D-X), (D′-I),(M-I)-(M-X), or (M′-I)) or a pharmaceutical composition thereof.

Moreover, methods described herein also include methods of protectingagainst or treating viral infection in a subject by administering to thesubject a conjugate described herein (e.g., a conjugate of any one offormulas (1)-(5), (D-I)-(D-X), (D′-I), (M-I)-(M-X), or (M′-I)). In someembodiments, the method further includes administering to the subject anantiviral agent or an antiviral vaccine.

Methods described herein also include methods of protecting against ortreating a viral infection in a subject by administering to said subject(1) a conjugate described herein (e.g., a conjugate of any one offormulas (1)-(5), (D-I)-(D-X), (D′-I), (M-I)-(M-X), or (M′-I)) and (2)an antiviral agent or an antiviral vaccine. Methods described hereinalso include methods of preventing, stabilizing, or inhibiting thegrowth of viral particles or preventing the replication or spread of avirus, by contacting the virus or a site susceptible to viral growthwith (1) a conjugate described herein (e.g., a conjugate of any one offormulas (1)-(5), (D-I)-(D-X), (D′-I), (M-I)-(M-X), or (M′-I)) and (2)an antiviral agent or an antiviral vaccine.

In some embodiments, the conjugate described herein (e.g., a conjugateof any one of formulas (1)-(5), (D-I)-(D-X), (D′-I), (M-I)-(M-X), or(M′-I)) is administered first, followed by administering of theantiviral agent or antiviral vaccine alone. In some embodiments, theantiviral agent or antiviral vaccine is administered first, followed byadministering of the conjugate described herein alone. In someembodiments, the conjugate described herein and the antiviral agent orantiviral vaccine are administered substantially simultaneously (e.g.,in the same pharmaceutical composition or in separate pharmaceuticalcompositions). In some embodiments, the conjugate described herein orthe antiviral agent or antiviral vaccine is administered first, followedby administering of the conjugate described herein and the antiviralagent or antiviral vaccine substantially simultaneously (e.g., in thesame pharmaceutical composition or in separate pharmaceuticalcompositions). In some embodiments, the conjugate described herein andthe antiviral agent or antiviral vaccine are administered firstsubstantially simultaneously (e.g., in the same pharmaceuticalcomposition or in separate pharmaceutical compositions), followed byadministering of the conjugate described herein or the antiviral agentor antiviral vaccine alone. In some embodiments, when a conjugatedescribed herein (e.g., a conjugate of any one of formulas (1)-(5),(D-I)-(D-X), (D′-I), (M-I)-(M-X), or (M′-I)) and an antiviral agent orantiviral vaccine are administered together (e.g., substantiallysimultaneously in the same or separate pharmaceutical compositions, orseparately in the same treatment regimen), inhibition of viralreplication of each of the conjugate and the antiviral agent orantiviral vaccine may be greater (e.g., occur at a lower concentration)than inhibition of viral replication of each of the conjugate and theantiviral agent or antiviral vaccine when each is used alone in atreatment regimen.

VIII. Pharmaceutical Compositions and Preparations

A conjugate described herein may be formulated in a pharmaceuticalcomposition for use in the methods described herein. In someembodiments, a conjugate described herein may be formulated in apharmaceutical composition alone. In some embodiments, a conjugatedescribed herein may be formulated in combination with an antiviralagent or antiviral vaccine in a pharmaceutical composition. In someembodiments, the pharmaceutical composition includes a conjugatedescribed herein (e.g., a conjugate described by any one of formulas(1)-(5), (D-I)-(D-X), (D′-I), (M-I)-(M-X), or (M′-I)) andpharmaceutically acceptable carriers and excipients.

Acceptable carriers and excipients in the pharmaceutical compositionsare nontoxic to recipients at the dosages and concentrations employed.Acceptable carriers and excipients may include buffers such asphosphate, citrate, HEPES, and TAE, antioxidants such as ascorbic acidand methionine, preservatives such as hexamethonium chloride,octadecyldimethylbenzyl ammonium chloride, resorcinol, and benzalkoniumchloride, proteins such as human serum albumin, gelatin, dextran, andimmunoglobulins, hydrophilic polymers such as polyvinylpyrrolidone,amino acid residues such as glycine, glutamine, histidine, and lysine,and carbohydrates such as glucose, mannose, sucrose, and sorbitol.

Examples of other excipients include, but are not limited to,antiadherents, binders, coatings, compression aids, disintegrants, dyes,emollients, emulsifiers, fillers (diluents), film formers or coatings,flavors, fragrances, glidants (flow enhancers), lubricants, sorbents,suspensing or dispersing agents, or sweeteners. Exemplary excipientsinclude, but are not limited to: butylated hydroxytoluene (BHT), calciumcarbonate, calcium phosphate (dibasic), calcium stearate,croscarmellose, crosslinked polyvinyl pyrrolidone, citric acid,crospovidone, cysteine, ethylcellulose, gelatin, hydroxypropylcellulose, hydroxypropyl methylcellulose, lactose, magnesium stearate,maltitol, mannitol, methionine, methylcellulose, methyl paraben,microcrystalline cellulose, polyethylene glycol, povidone,pregelatinized starch, propyl paraben, retinyl palmitate, shellac,silicon dioxide, sodium carboxymethyl cellulose, sodium citrate, sodiumstarch glycolate, sorbitol, starch (corn), stearic acid, stearic acid,sucrose, talc, titanium dioxide, vitamin A, vitamin E, vitamin C, andxylitol.

The conjugates herein may have ionizable groups so as to be capable ofpreparation as pharmaceutically acceptable salts. These salts may beacid addition salts involving inorganic or organic acids or the saltsmay, in the case of acidic forms of the conjugates herein be preparedfrom inorganic or organic bases. Frequently, the conjugates are preparedor used as pharmaceutically acceptable salts prepared as additionproducts of pharmaceutically acceptable acids or bases. Suitablepharmaceutically acceptable acids and bases are well-known in the art,such as hydrochloric, sulphuric, hydrobromic, acetic, lactic, citric, ortartaric acids for forming acid addition salts, and potassium hydroxide,sodium hydroxide, ammonium hydroxide, caffeine, various amines, and thelike for forming basic salts. Methods for preparation of the appropriatesalts are well-established in the art.

Representative acid addition salts include, but are not limited to,acetate, adipate, alginate, ascorbate, aspartate, benzenesulfonate,benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate,citrate, cyclopentanepropionate, digluconate, dodecylsulfate,ethanesulfonate, fumarate, glucoheptonate, glycerophosphate,hemisulfate, heptonate, hexanoate, hydrobromide, hydrochloride,hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate,lauryl sulfate, malate, maleate, malonate, methanesulfonate,2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate,pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate,pivalate, propionate, stearate, succinate, sulfate, tartrate,thiocyanate, toluenesulfonate, undecanoate, and valerate salts.Representative alkali or alkaline earth metal salts include, but are notlimited to, sodium, lithium, potassium, calcium, and magnesium, as wellas nontoxic ammonium, quaternary ammonium, and amine cations, including,but not limited to ammonium, tetramethylammonium, tetraethylammonium,methylamine, dimethylamine, trimethylamine, triethylamine, andethylamine.

Depending on the route of administration and the dosage, a conjugateherein or a pharmaceutical composition thereof used in the methodsdescribed herein will be formulated into suitable pharmaceuticalcompositions to permit facile delivery. A conjugate (e.g., a conjugateof any one of formulas (1)-(5), (D-I)-(D-X), (D′-I), (M-I)-(M-X), or(M′-I)) or a pharmaceutical composition thereof may be formulated to beadministered intramuscularly, intravenously (e.g., as a sterile solutionand in a solvent system suitable for intravenous use), intradermally,intraarterially, intraperitoneally, intralesionally, intracranially,intraarticularly, intraprostatically, intrapleurally, intratracheally,intranasally, intravitreally, intravaginally, intrarectally, topically,intratumorally, peritoneally, subcutaneously, subconjunctival,intravesicularlly, mucosally, intrapericardially, intraumbilically,intraocularally, orally (e.g., a tablet, capsule, caplet, gelcap, orsyrup), topically (e.g., as a cream, gel, lotion, or ointment), locally,by inhalation, by injection, or by infusion (e.g., continuous infusion,localized perfusion bathing target cells directly, catheter, lavage, incremes, or lipid compositions). Depending on the route ofadministration, a conjugate herein or a pharmaceutical compositionthereof may be in the form of, e.g., tablets, capsules, pills, powders,granulates, suspensions, emulsions, solutions, gels including hydrogels,pastes, ointments, creams, plasters, drenches, osmotic delivery devices,suppositories, enemas, injectables, implants, sprays, preparationssuitable for iontophoretic delivery, or aerosols. The compositions maybe formulated according to conventional pharmaceutical practice.

A conjugate described herein may be formulated in a variety of ways thatare known in the art. For use as treatment of human and animal subjects,a conjugate described herein can be formulated as pharmaceutical orveterinary compositions. Depending on the subject (e.g., a human) to betreated, the mode of administration, and the type of treatment desired,e.g., prophylaxis or therapy, a conjugate described herein is formulatedin ways consonant with these parameters. A summary of such techniques isfound in Remington: The Science and Practice of Pharmacy, 22nd Edition,Lippincott Williams & Wilkins (2012); and Encyclopedia of PharmaceuticalTechnology, 4th Edition, J. Swarbrick and J. C. Boylan, Marcel Dekker,New York (2013), each of which is incorporated herein by reference.

Formulations may be prepared in a manner suitable for systemicadministration or topical or local administration. Systemic formulationsinclude those designed for injection (e.g., intramuscular, intravenousor subcutaneous injection) or may be prepared for transdermal,transmucosal, or oral administration. The formulation will generallyinclude a diluent as well as, in some cases, adjuvants, buffers, andpreservatives. The conjugates can be administered also in liposomalcompositions or as microemulsions. Systemic administration may alsoinclude relatively noninvasive methods such as the use of suppositories,transdermal patches, transmucosal delivery and intranasaladministration. Oral administration is also suitable for conjugatesherein. Suitable forms include syrups, capsules, and tablets, as isunderstood in the art.

The pharmaceutical compositions can be administered parenterally in theform of an injectable formulation. Pharmaceutical compositions forinjection can be formulated using a sterile solution or anypharmaceutically acceptable liquid as a vehicle. Formulations may beprepared as solid forms suitable for solution or suspension in liquidprior to injection or as emulsions. Pharmaceutically acceptable vehiclesinclude, but are not limited to, sterile water, physiological saline,and cell culture media (e.g., Dulbecco's Modified Eagle Medium (DMEM),α-Modified Eagles Medium (α-MEM), F-12 medium). Such injectablecompositions may also contain amounts of nontoxic auxiliary substancessuch as wetting or emulsifying agents, pH buffering agents, such assodium acetate and sorbitan monolaurate. Formulation methods are knownin the art, see e.g., Pharmaceutical Preformulation and Formulation, 2ndEdition, M. Gibson, Taylor & Francis Group, CRC Press (2009).

The pharmaceutical compositions can be prepared in the form of an oralformulation. Formulations for oral use include tablets containing theactive ingredient(s) in a mixture with non-toxic pharmaceuticallyacceptable excipients. These excipients may be, for example, inertdiluents or fillers (e.g., sucrose, sorbitol, sugar, mannitol,microcrystalline cellulose, starches including potato starch, calciumcarbonate, sodium chloride, lactose, calcium phosphate, calcium sulfate,or sodium phosphate); granulating and disintegrating agents (e.g.,cellulose derivatives including microcrystalline cellulose, starchesincluding potato starch, croscarmellose sodium, alginates, oralginicacid); binding agents (e.g., sucrose, glucose, sorbitol, acacia, alginicacid, sodium alginate, gelatin, starch, pregelatinized starch,microcrystalline cellulose, magnesium aluminum silicate,carboxymethylcellulose sodium, methylcellulose, hydroxypropylmethylcellulose, ethylcellulose, polyvinylpyrrolidone, or polyethyleneglycol); and lubricating agents, glidants, and antiadhesives (e.g.,magnesium stearate, zinc stearate, stearic acid, silicas, hydrogenatedvegetable oils, or talc). Formulations for oral use may also be providedas chewable tablets, or as hard gelatin capsules wherein the activeingredient is mixed with an inert solid diluent (e.g., potato starch,lactose, microcrystalline cellulose, calcium carbonate, calciumphosphate or kaolin), or as soft gelatin capsules wherein the activeingredient is mixed with water or an oil medium, for example, peanutoil, liquid paraffin, or olive oil. Powders, granulates, and pellets maybe prepared using the ingredients mentioned above under tablets andcapsules in a conventional manner using, e.g., a mixer, a fluid bedapparatus or a spray drying equipment.

Other pharmaceutically acceptable excipients for oral formulationsinclude, but are not limited to, colorants, flavoring agents,plasticizers, humectants, and buffering agents. Formulations for oraluse may also be provided as chewable tablets, or as hard gelatincapsules wherein the active ingredient is mixed with an inert soliddiluent (e.g., potato starch, lactose, microcrystalline cellulose,calcium carbonate, calcium phosphate or kaolin), or as soft gelatincapsules wherein the active ingredient is mixed with water or an oilmedium, for example, peanut oil, liquid paraffin, or olive oil. Powders,granulates, and pellets may be prepared using the ingredients mentionedabove under tablets and capsules in a conventional manner using, e.g., amixer, a fluid bed apparatus or a spray drying equipment.

Dissolution or diffusion controlled release of a conjugate describedherein (e.g., a conjugate of any one of formulas (1)-(5), (D-I)-(D-X),(D′-I), (M-I)-(M-X), or (M′-I)) or a pharmaceutical composition thereofcan be achieved by appropriate coating of a tablet, capsule, pellet, orgranulate formulation of the conjugate, or by incorporating theconjugate into an appropriate matrix. A controlled release coating mayinclude one or more of the coating substances mentioned above and/or,e.g., shellac, beeswax, glycowax, castor wax, carnauba wax, stearylalcohol, glyceryl monostearate, glyceryl distearate, glycerolpalmitostearate, ethylcellulose, acrylic resins, dl-polylactic acid,cellulose acetate butyrate, polyvinyl chloride, polyvinyl acetate, vinylpyrrolidone, polyethylene, polymethacrylate, methylmethacrylate,2-hydroxymethacrylate, methacrylate hydrogels, 1,3 butylene glycol,ethylene glycol methacrylate, and/or polyethylene glycols. In acontrolled release matrix formulation, the matrix material may alsoinclude, e.g., hydrated methylcellulose, carnauba wax and stearylalcohol, carbopol 934, silicone, glyceryl tristearate, methylacrylate-methyl methacrylate, polyvinyl chloride, polyethylene, and/orhalogenated fluorocarbon.

The pharmaceutical composition may be formed in a unit dose form asneeded. The amount of active component, e.g., a conjugate describedherein (e.g., a conjugate of any one of formulas (1)-(5), (D-l)-(D-X),(D′-I), (M-I)-(M-X), or (M′-I)), included in the pharmaceuticalcompositions are such that a suitable dose within the designated rangeis provided (e.g., a dose within the range of 0.01-100 mg/kg of bodyweight).

IX. Routes of Administration and Dosages

In any of the methods described herein, conjugates herein may beadministered by any appropriate route for treating or protecting againsta viral infection (e.g., an influenza infection), or for preventing,stabilizing, or inhibiting the proliferation or spread of a virus (e.g.,an influenza virus). Conjugates described herein may be administered tohumans, domestic pets, livestock, or other animals with apharmaceutically acceptable diluent, carrier, or excipient. In someembodiments, administering comprises administration of any of theconjugates described herein (e.g., conjugates of any one of formulas(1)-(5), (D-I)-(D-X), (D′-I), (M-I)-(M-X), or (M′-I)) or compositionsintramuscularly, intravenously (e.g., as a sterile solution and in asolvent system suitable for intravenous use), intradermally,intraarterially, intraperitoneally, intralesionally, intracranially,intraarticularly, intraprostatically, intrapleurally, intratracheally,intranasally, intravitreally, intravaginally, intrarectally, topically,intratumorally, peritoneally, subcutaneously, subconjunctival,intravesicularlly, mucosally, intrapericardially, intraumbilically,intraocularally, orally (e.g., a tablet, capsule, caplet, gelcap, orsyrup), topically (e.g., as a cream, gel, lotion, or ointment), locally,by inhalation, by injection, or by infusion (e.g., continuous infusion,localized perfusion bathing target cells directly, catheter, lavage, incremes, or lipid compositions). In some embodiments, if an antiviralagent is also administered in addition to a conjugate described herein,the antiviral agent or a pharmaceutical composition thereof may also beadministered in any of the routes of administration described herein.

The dosage of a conjugate described herein (e.g., a conjugate of any oneof formulas (1)-(5), (D-I)-(D-X), (D′-I), (M-I)-(M-X), or (M′-I)) orpharmaceutical compositions thereof depends on factors including theroute of administration, the disease to be treated (e.g., the extentand/or condition of the viral infection), and physical characteristics,e.g., age, weight, general health, of the subject. Typically, the amountof the conjugate or the pharmaceutical composition thereof containedwithin a single dose may be an amount that effectively prevents, delays,or treats the viral infection without inducing significant toxicity. Apharmaceutical composition may include a dosage of a conjugate describedherein ranging from 0.01 to 500 mg/kg (e.g., 0.01, 0.1, 0.2, 0.3, 0.4,0.5, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 150, 200,250, 300, 350, 400, 450, or 500 mg/kg) and, in a more specificembodiment, about 0.1 to about 30 mg/kg and, in a more specificembodiment, about 1 to about 30 mg/kg. In some embodiments, when aconjugate described herein (e.g., a conjugate of any one of formulas(1)-(5), (D-I)-(D-X), (D′-I), (M-I)-(M-X), or (M′-I)) and an antiviralagent or antiviral vaccine are administered in combination (e.g.,substantially simultaneously in the same or separate pharmaceuticalcompositions, or separately in the same treatment regimen), the dosageneeded of the conjugate described herein may be lower than the dosageneeded of the conjugate if the conjugate was used alone in a treatmentregimen.

A conjugate described herein (e.g., a conjugate of any one of formulas(1)-(5), (D-I)-(D-X), (D′-I), (M-I)-(M-X), or (M′-I)) or apharmaceutical composition thereof may be administered to a subject inneed thereof, for example, one or more times (e.g., 1-10 times or more;1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times) daily, weekly, monthly,biannually, annually, or as medically necessary. Dosages may be providedin either a single or multiple dosage regimens. The timing betweenadministrations may decrease as the medical condition improves orincrease as the health of the patient declines. The dosage and frequencyof administration may be adapted by the physician in accordance withconventional factors such as the extent of the infection and differentparameters of the subject.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a description of how the compositions and methodsdescribed herein may be used, made, and evaluated, and are intended tobe purely exemplary of the invention and are not intended to limit thescope of what the inventors regard as their invention.

Example 1: Preparation of Fc Constructs

Reverse translations of the amino acids comprising the proteinconstructs (SEQ ID NOs: 1, 3, 5, 7, 9, 12, and 14) were synthesized bysolid-phase synthesis. The oligonucleotide templates were cloned intopcDNA3.1 (Life Technologies, Carlsbad, Calif., USA) at the cloning sitesBamHI and XhoI (New England Biolabs, Ipswich, Mass., USA) and includedsignal sequences derived from the human Interleukin-2 or human albumin.The pcDNA3.1 plasmids were transformed into Top10 E. coli cells(LifeTech). DNA was amplified, extracted, and purified using thePURELINK® HiPure Plasmid Filter Maxiprep Kit (LifeTech). The plasmid DNAis delivered, using the ExpiFectamine™ 293 Transfection Kit (LifeTech),into HEK-293 cells per the manufacturer's protocol. Cells werecentrifuged, filtered, and the supernatants were purified usingMabSelect Sure Resin (GE Healthcare, Chicago, Ill., USA). Purifiedmolecules were analyzed using 4-12% Bis Tris SDS PAGE gels by loading1-2 μg of each molecule into the gel, and staining using instant Bluestaining. Each gel included a molecular weight ladder with the indicatedmolecular weight standards (FIGS. 2-8). Reduced and non-reduced lanesare denoted by “R” and “NR”. FIGS. 2-8 show non-reducing and reducingSDS-PAGE of an Fc domain formed from Fc domain monomers having thesequences of SEQ ID NOs: 1, 3, 5, 7, 9, 12, and 14, respectively.

Example 2. Synthesis of Zanamivir Intermediate

Step a

Methyl5-acetamido-7,8,9-O-triacetyl-2,6-anhydro-4-azido-3,4,5-trideoxy-D-glycero-D-galacto-non-2-enonate(4.56 g, 10.0 mmol) was dissolved in anhydrous THF (15 mL), and thesolution was cooled to approximately 13° C. Triphenylphosphine (2.89 g,11 mmol) was added in portions over 20 minutes. The resulting mixturewas stirred at approximately 13° C. to room temperature for 2 hours,then a solution of LiOH (24 mg, 1 mmol) in water (1.5 mL) was addeddropwise. After stirring for 28 hours, the reaction mixture was addedwith N,N′-bis-boc-1-guanylpyrazole (3.26 g, 10.5 mmol) and4-dimethylaminopyridine (244.4 mg, 2 mmol). The reaction was stirred for1.5 days. It was then diluted with a 1:1 mixture of ethylacetate:hexanes (100 mL) and extracted with water (30 mL). The aqueouslayer was back-extracted with ethyl acetate (30 mL). The combinedorganic layers were concentrated by rotary evaporation. The residue waspurified through C18 reversed phase column chromatography (150 g, 25 to70% acetonitrile and water). The collected fractions were concentratedby rotary evaporation at room temperature. A cloudy aqueous solutionresulted and the majority of the product deposited on the flask as agel. The solution was then extracted with ethyl acetate (150 mL). Theorganic layer was used to re-dissolve the gel material. It was thendried over Na₂SO₄, concentrated by rotary evaporation, and further driedunder high vacuum to afford the title compound as a white foam. Yield6.24 g, 92.8%. Ion found by LCMS: [M+H]⁺=673.2.

Step b.

A solution of the product from step-a (6.24 g, 9.28 mmol) in anhydrousMeOH (20 mL) was cooled in an ice-water bath and a 0.5 M solution ofsodium methoxide in MeOH (26 mL, 13 mmol) was slowly added. The reactionwas stirred for 1 hour, then its pH was carefully adjusted to 7 to 7.5by dropwise addition of a 4 N solution of HCl in dioxane (3 mL). Thesolvent was removed by rotary evaporation at a temperature not greaterthan room temperature. The residue was diluted with a 2:1 mixture ofethyl acetate and hexanes (150 mL), and the resulting solution wasextracted with water (20 mL). The aqueous layer was back-extracted withethyl acetate (30 mL). The combined organic layers were dried overNa₂SO₄, concentrated by rotary evaporation, and further dried under highvacuum. The product was carried to the subsequent step without furtherpurification. Yield 5.03 g, 99.2%. Ion found by LCMS: [M+H]⁺=547.2.

Step c

The step-b product (713 mg, 1.3 mmol) in anhydrous DCM (6 mL) was cooledin an ice-water bath and 4-dimethylaminopyridine (159.8 mg, 1.3 mmol)and DIPEA (520 mg, 4 mmol) were added. The mixture was then dropwiseadded with a solution of 4-nitrophenyl chloroformate (356.6 mg, 2.2mmol) in anhydrous DCM (2 mL). The ice-water bath was then removed, andthe reaction mixture was stirred for 3 hours and monitored by LCMS(additional 4-nitrophenyl chloroformate may be added if needed). Afterthe reaction was complete, it was quenched with water (10 mL), and theorganic layer was extracted and concentrated by rotary evaporation. Theresidue was purified by C18 reversed phase column chromatography (100 g,20 to 70% acetonitrile and water). Acetonitrile in the collectedfractions was removed by rotary evaporation at room temperature. Theaqueous layer was then extracted with a 1:1 mixture of ethyl acetate andhexane (120 mL). The aqueous layer was back-extracted with ethyl acetate(30 mL). The combined organic layers were dried over Na₂SO₄,concentrated by rotary evaporation, and further dried under high vacuumto afford the title compound as a white solid. Yield 520 mg, 70%. Ionfound by LCMS: [M+H]⁺=573.2.

Example 3. Synthesis of Linker-1

Step a

To a mixture of Z-D-glutamic γ-methyl ester (2.0 g, 6.77 mmol) andglycine methyl ester HCl (1.282 g, 10.2 mmol) in anhydrous DMF (7 mL)was added DIPEA (2.02 g, 15.57 mmol) followed by HATU (2.66 g, 7.0 mmol)in portions over 25 minutes. After HATU was dissolved, an additionalamount of DIPEA (1.15 g, 8.8 mmol) was added. The reaction mixture wasstirred for 1.5 hours, then extracted with 5% aqueous HCl (100 mL) andEtOAc (100 mL×2). The organic layer was concentrated by rotaryevaporation. The residue was re-extracted with water (100 mL) andEtOAc/hexanes (2:1, 150 mL). The organic layer was dried over Na₂SO₄,concentrated by rotary evaporation and further dried under high vacuumto a white solid. The crude product was carried on to the subsequentstep without further purification. Yield 2.3 g, 93%. Ion found by LCMS:[M+H]⁺=367.

Step b

The step-a product (2.3 g, 6.29 mmol) was dissolved in a 1:1 mixture ofMeOH and THF (10 mL). After the solution was cooled in an ice-waterbath, a LiOH monohydrate (630 mg, 15 mmol) solution in water (9 mL) wasadded in portions over 1.5 hours. After stirring for 2 additional hours,the reaction mixture was neutralized with 4N HCl in dioxane (3.7 mL).The organic solvent was partially removed by rotary evaporation at roomtemperature. The remaining material was directly purified by RPLC (150g, 0 to 39% acetonitrile and water). Yield 2.03 g, 88.7% over two steps.Ion found by LCMS: [M+H]⁺=339.2.

Step c

To a mixture of the step-b product (1.41 g, 4.17 mmol) andN-Boc-1,6-diaminohexane (1.99 g, 9.2 mmol) in anhydrous DMF (6 mL) andDIPEA (1.3 g, 10 mmol), a solution of HATU (3.5 g, 9.2 mmol) in DMF (10mL) was added byway of syringe pump at a rate of 11 mL/hr. Upon completeaddition of the HATU, the reaction was stirred for 30 more minutes anddirectly purified by RPLC (150 g, 10 to 50% acetonitrile and water,using 0.1% TFA as modifier). Yield 1.3 g, 42.4%. Ion found by LCMS:[M+H]⁺=735, [M-Boc+H]+=635.4.

Step d

The step-c product (1.3 g, 1.77 mmol) was dissolved in MeOH (20 mL), andPd/C was added to the solution. The mixture was stirred under hydrogenfor 4 hours. Pd/C was filtered off, and the filtrate was concentrated byrotary evaporation and further dried under high vacuum. Yield 1.03 g,96.9%. Ion found by LCMS: [M+H]⁺=601.

Step e

A flamed-dried reaction flask was flushed with nitrogen and charged with4-azidobutyric acid (77 mg, 0.6 mmol), N-hydroxy succinimide (92 mg, 0.8mmol) and anhydrous DMF (0.5 mL). The mixture was stirred to dissolvethe solids, and then DCC (125.5 mg, 0.608 mmol) was added. Afterstirring for one hour, the step-d product (300 mg, 0.5 mmol) was addedto the reaction mixture. The reaction was stirred for 6 hours anddirectly purified using RPLC (150 g, 10 to 70% acetonitrile and water,using 0.1% TFA as modifier). The collected fractions were lyophilized toa white solid (LCMS: [M+H]⁺=712, [M-Boc+H]⁺=612). The material wasre-dissolved in DCM (˜2 mL) and TFA (˜1 mL) and stirred for 15 minutes.It was then concentrated by rotary evaporation, and the residue waspurified by RPLC (50 g, 0 to 40% acetonitrile and water). Yield 255 mg,68.9%. Ions found by LCMS: [M+H]⁺=512, [(M+2H)/2]⁺=256.

Example 4. Synthesis of Int-1

Step a

To a solution of the Zanamivir intermediate (Example 2) (532.5 mg, 0.93mmol) in anhydrous THF (2 mL) was added DMAP (490 mg, 4 mmol), followedby bis(pentafluorophenyl)carbonate (385 mg, 0.911 mmol). After stirringovernight, a solution of Linker-1 (Example 3) (245.6 mg, 0.332 mmol) inanhydrous DMF (1 mL) and DIPEA (91 mg, 0.3 mmol) was added to thereaction mixture. The reaction was continued for 2 hours, then purifiedby RPLC (100 g, 5 to 67% acetonitrile and water). Yield 135 mg, 23.8%.Ion found by LCMS: [(M+2H)/2]⁺=845.9.

Step b

The step-a product (135 mg, 0.079 mmol) was dissolved in TFA (0.5 mL),and the solution was stirred at room temperature for 20 minutes. It wasthen directly purified by RPLC (50 g, 5 to 32% acetonitrile and water).Yield 88 mg, 85.1%. Ion found by LCMS: [(M+2H)/2]⁺=654.8.

Step c

A solution of the step-b product (88 mg, 0.0673 mmol) in MeOH (1.5 mL)was cooled in an ice-water bath and LiOH (5 mg, 0.2 mmol) in water (0.5mL) was added. After the mixture was stirred for 5 hours, it wasacidified with 4N HCl solution in dioxane (0.1 mL) and purified by HPLC(5 to 20% acetonitrile and water, using 0.1% TFA as modifier). Yield76.4 mg, 78%. Ion found by LCM: [(M+2H)/2]⁺=614.8.

Example 5. Synthesis of Linker-2

Step a

To a solution of propargyl-PEG4-acid (364.4 mg, 1.4 mmol) in anhydrousDMF (2 mL) was added HATU (558.9 mg, 1.47 mmol). After stirring todissolve all the coupling reagent, DIPEA (390 mg, 3 mmol) was added andstirred for 10 minutes. A solution of the Linker-1 step-d product(Example 3, step d) (701.1 mg, 1.167 mmol) in anhydrous DMF (1 mL) wasadded. The resulting mixture was stirred for 30 minutes and directlypurified by RPLC (100 g, 5 to 60% acetonitrile and water). Yield 830 mg,84.4%. Ion found by LCMS: [M+H]⁺=843.

Step b

The step-a product was dissolved in THF (5 mL) and treated with 4N HClsolution in dioxane (2.5 mL). After stirring at room temperatureovernight, the reaction mixture was concentrated by rotary evaporation.The residue was re-dissolved in acetonitrile/water (1:1, 16 mL), and thesolution was lyophilized. The crude product was carried on to thesubsequent step without further purification. Yield 761.8 mg, 100%. Ionfound by LCMS: [M+H]⁺=643.8.

Example 6. Synthesis of Int-2

Step a

A flame-dried reaction flask was flushed with nitrogen and charged withZanamivir intermediate (Example 2) (533.6 mg, 0.812 mmol), DMAP (99.8mg, 0.81 mmol), and anhydrous DCM (1 mL). After stirring to dissolve thestarting material, the solution was cooled in an ice-water bath and4-nitrophenyl chloroformate (242 mg, 1.2 mmol) was added. The resultingmixture was stirred for 5 hours, then added into a solution of theLinker-2 (Example 5) (228.4 mg, 0.319 mmol) in anhydrous DMF (1 mL) andDIPEA (130 mg, 1 mmol). The reaction was stirred overnight and purifiedby RPLC (150 g, 20 to 65% acetonitrile and water, using 0.1% TFA asmodifier). The collected fractions were lyophilized. Yield 178.3 mg,30.4%. Ion found by LCMS: [(M+2H)/2]⁺=920.5, [(M+3H)/3]⁺=614.2.

Step b

The step-a product (178.3 mg, 0.0969 mmol) was dissolved in TFA (0.5mL). The solution was stirred for 20 minutes, then directly purified byHPLC (0 to 25% acetonitrile and water, using 0.1% TFA as modifier).Yield 91.8 mg, 56.8%. Ions found by LCMS: [(M+2H)/2]⁺=720.4,[(M+3H)/3]⁺=480.6.

Step c

The step-b product (91.8 mg, 0.055 mmol) was dissolved in MeOH (1 mL),and the solution was cooled in an ice-water bath. LiOH monohydrate (21mg, 0.5 mmol) in water (1 mL) was added in portions over 1 hour. Afterstirring for 2 more hours, the reaction mixture was acidified with a 4NHCl solution in dioxane (0.3 mL) and purified by HPLC (0 to 20%acetonitrile and water, using 0.1% TFA as modifier). Yield 36.2 mg,59.4%.%. Ions found by LCMS: [(M+2H)/2]⁺=680.3, [(M+3H)/3]⁺=454.0.

Example 7. Synthesis of h-IgG1 Fc-PEG4-azide

PEG4-azidoNHS ester (98%, 180 μmol, 9.5 equivalents, 71.4 mg in 0.5 mLof DMF and diluted to 3.60 mL with pH 7.4 PBS 1× buffer solution) wasadded to a solution of h-IgG1 Fc (SEQ ID NO: 4) (1103 mg in 70.0 mL ofpH 7.4 PBS, MW58,000 Da, 19.0 μmol) and the mixture was shaken gentlyfor 12 hours at ambient temperature. The solution was concentrated usinga centrifugal concentrator (30,000 MWCO) to a volume of ˜1.5 mL. Thecrude mixture was diluted 1:10 in PBS pH 7.4, and concentrated again.This wash procedure was repeated for total of three times. The smallmolecule reagent was removed with this wash procedure. The concentratedFc-PEG4-azide was diluted to 70.0 mL with pH 7.4 PBS 1× buffer and readyfor Click conjugation. The purified material was quantified using aNanodrop™ UV visible spectrophotomer (using a calculated extinctioncoefficient based on the amino acid sequence of h-IgG1). Yield isquantitative after purification. DAR=4.3 determined by MALDI. The DARvalue can be adjusted by altering the equivalents of PEG4-azido NHSester in near linear relation. For example, when 7.0 equivalents ofPEG4-azide NHS ester is used the DAR value will be at 3.0.

Example 8. Synthesis of Recombinant Mouse Serum Albumin (MSA)-PEG4-azide

PEG4-azidoNHS ester (98%, 81.7 μmol, 4.5 equivalents, 32.4 mg in 0.3 mLof DMF and diluted to 1.63 mL with pH 7.4 PBS 1× buffer solution) wasadded to a solution of recombinant mouse serum albumin (SEQ ID NO: 71)(1200 mg in 75.0 mL of pH 7.4 PBS, MW66,000 Da, 18.2 μmol) and themixture was shaken gently for 12 hours at ambient temperature. Thesolution was concentrated using a centrifugal concentrator (30,000 MWCO)to a volume of 1.5 mL. The crude mixture was diluted 1:10 in PBS pH 7.4,and concentrated again. This wash procedure was repeated for total ofthree times. The small molecule reagent was removed with this washprocedure. The concentrated MSA-PEG4-azide was diluted to 75.0 mL withpH 7.4 PBS 1× buffer and ready for Click conjugation. The purifiedmaterial was quantified using a Nanodrop™ UV visible spectrophotomer(using a calculated extinction coefficient based on the amino acidsequence of h-IgG1). Yield is quantitative after purification. DAR=3.5determined by MALDI. The DAR value can be adjusted by altering theequivalents of PEG4-azido NHS ester similar to h-IgG1 Fc (Example 7).

Example 9. Synthesis of Conjugate 1

A PBS solution of h-IgG1 Fc-PEG4-azide (Example 7) (50 mg, 2.815 mL,0.8591 μmol) was added into a 15 mL centrifuge tube containing Int-2(Example 6) (35.2 mg, 0.02217 mmol). After the mixture was gently shakento dissolve all Int-2, 344 μl of a solution of L-ascorbic acid sodium(59.4 mg, 0.3 mmol), copper (II) sulfate (10 mg, 0.05 mmol), and THPTA(23 mg, 0.05 mmol) in PBS 7.4 buffer (1 mL) was added. The resultingmixture was gently shaken overnight. It was purified by affinitychromatography over a protein A column, followed by size exclusionchromatography as described in Example 8. Maldi TOF analysis of thepurified final product gave an average mass of 63797 Da (DAR=3.4). Yield27.39 mg, 55% yield. FIG. 9 shows a non-reducing SDS-PAGE of Conjugate1.

Example 10. Purification of Conjugates

Crude mixture was diluted 1:10 in PBS pH 7.4, and purified usingMabSelect Sure Resin (GE Healthcare, Chicago, Ill., USA), followed bysize exclusion chromatography. (HiLoad 26/600 Superdex200 pg, GEHealthcare, Chicago, Ill., USA). Fractions containing purified conjugatewere pooled and concentrated to approximately 20 mg/mL using acentrifugal concentrator (30,000 MWCO). Purified material was quantifiedusing a Nanodrop™ UV visible spectrophotomer using a calculatedextinction coefficient based on the amino acid sequence of hIgG1Fc(myc). Purified molecules were analyzed using 4-12% Bis Tris SDS PAGEgels by loading 1 μg of each molecule into the gel, and staining usingInstant Blue (Expedeon, San Diego, Calif., USA). Each gel included amolecular weight ladder with the indicated molecular weight standards.Yields were calculated and purity determined by Agilent Analytical HPLC.Product peak and MW were found by Maldi MS and a final DAR calculated.

Example 11. Synthesis of Int-3

Step a

To a solution of propargyl-PEG4-acid (260 mg, 1 mmol) and HATU (380.2mg, 1 mmol) in anhydrous DMF (1 mL) was added DIPEA (130 mg). Afterstirring 5 minutes, NH-bis(PEG3-Boc) (500 mg, 0.881 mmol) was added andstirring was continued at room temperature overnight. It was thendirectly purified by RPLC 9100 g, 5 to 50% acetonitrile and water, using0.1% TFA as modifier). Yield 683 mg, 95.8%. Ions found by LCMS:[M+H]⁺=810.4, [M-Boc+H]⁺=710.4, [(M-2Boc+2H)/2]⁺=305.8.

Step b

The step-a product was dissolved in TFA (1 mL). The solution was stirredfor 2 hours and then directly purified through RPLC (100 g, 0 to 30%acetonitrile and water). Yield 589 mg, 98.7%. Ion found by LCMS:[(M+2H)/2]⁺=305.8.

Step c

A flame-dried reaction flask was flushed with nitrogen and charged withZanamivir intermediate (Example 2) (572 mg, 1 mmol) and anhydrous DCM (1mL). After stirring to dissolve the starting material, the solution wascooled in an ice-water bath and 4-nitrophenyl chloroformate (302.4 mg,1.5 mmol) was added followed by DMAP (22.4 mg, 0.2 mmol). The resultingmixture was stirred for 5 hours, then quenched water (0.2 mL) was added.After stirring for 10 minutes, the step-b product (256.7 mg, 0.355 mmol)in anhydrous DMF (1 mL) and DIPEA (163.8 mg, 1.26 mmol) was added.Stirring was continued for 2 hours and then the reaction was directlypurified by RPLC (150 g, 20 to 65% acetonitrile and water, using 0.1%TFA as modifier). The collected fractions were lyophilized. Yield 422.8mg of the desired product which was contaminated with some impurities,<69% yield. The material was carried on to the subsequent step withoutfurther purification. Ion found by LCMS: [(M+2H)/2]⁺=903.9,[(M+3H)/3]⁺=603.2.

Step d

The step-c product (422.8 mg, <0.245 mmol) was dissolved in TFA (1 mL).The solution was stirred for 20 minutes, then directly purified by HPLC(5 to 25% acetonitrile and water, using 0.1% TFA as modifier). Yield169.7 mg, 29.2% over two steps. Ions found by LCMS: [(M+2H)/2]⁺=704.0,[(M+3H)/3]⁺=469.6.

Step e

The step-d product (169.7 mg, 0.0923 mmol) was dissolved in MeOH (1.5mL), and the solution was cooled in an ice-water bath. LiOH monohydrate(21 mg, 0.5 mmol) in water (1 mL) was added in portions over 1 hour.After stirring overnight, the reaction mixture was acidified with Dowex50W×8 hydrogen form and purified through RPLC (0 to 30% acetonitrile andwater, using 0.1% TFA as modifier). Yield 107.9 mg, 66.9%. Ions found byLCMS: [(M+2H)/2]⁺=663.8, [(M+3H)/3]⁺=442.9.

Example 12. Synthesis of Conjugate 2

The title conjugate was prepared analogously to Conjugate 1 (Example 9)using Int-3 (Example 11). Maldi TOF analysis of the purified finalproduct gave an average mass of 63561 Da (DAR=3.3). Yield 43.4 mg, 43%yield. FIG. 10 shows a non-reducing SDS-PAGE of Conjugate 2.

Example 13. Synthesis of InM

Step a

A flame-dried reaction flask was flushed with nitrogen and charged withZanamivir Intermediate (Example 2) (343.2 mg, 0.6 mmol) and anhydrousDCM (1.5 mL). The solution was cooled in an ice-water bath and addedwith DIPEA (234 mg, 1.8 mmol) followed by 4-nitrophenyl chloroformate(121 mg, 0.6 mmol) and DMAP (67.4 mg, 0.6 mmol). The resulting mixturewas stirred for 15 minutes, then added with an additional amount of4-nitrophenyl chloroformate (121 mg, 0.6 mmol). After stirring for 2hours, water (0.2 mL) was added to quench unreacted chloroformate. Afterstirred for 10 minutes, the reaction mixture was added with a solutionof propargyl-PEG4-amine (185 mg, 0.8 mmol) in anhydrous DMF (0.5 mL).The reaction was continued for 1 hour and then directly purified throughRPLC (100 g, 5 to 60% acetonitrile and water, using 0.1% TFA asmodifier). The collected fractions were lyophilized. Yield 355 mg,71.3%. Ion found by LCMS: [M+H]⁺=830.2.

Step b

The step-a product (355 mg, 0.428 mmol) was dissolved in TFA (1 mL). Thesolution was stirred overnight, then directly purified by RPLC (5 to 25%acetonitrile and water, using 0.1% TFA as modifier). Yield 260.2 mg,70.9% over two steps. Ion found by LCMS: [M+H]⁺=630.2.

Step c

The step-b product (260.2 mg, 0.303 mmol) was dissolved in MeOH (1.5mL). After the solution was cooled in an ice-water bath, a solution ofLiOH monohydrate (42 mg, 1 mmol) in water (1 mL) was added in portionsover 1 hour. The reaction was stirred overnight, then acidified withDowex 50W×8 hydrogen form and purified by RPLC (0 to 50% acetonitrileand water, using 0.1% TFA as modifier). Yield 78.1 mg, 99%. Ions foundby LCMS: [M+H]⁺=590.2.

Example 14. Synthesis of Conjugate 3

The title conjugate was prepared analogously to Conjugate 1 (Example 9)using Int-4 (Example 13). Maldi TOF analysis of the purified finalproduct gave an average mass of 61182 Da (DAR=3.4). Yield 50.89 mg, 51%yield. FIG. 11 shows a non-reducing SDS-PAGE of Conjugate 3.

Example 15. Synthesis of Int-5

Step a

A flame-dried reaction flask was flushed with nitrogen and charged with(1S,2S,3R,4R)-methyl3-((S)-1-acetamido-2-ethylbutyl)-4-(tert-butoxycarbonylamino)-2-hydroxycyclopentanecarboxylate(320.4 mg, 0.8 mmol) and anhydrous DCM (2 mL). The solution was cooledin an ice-water bath and DIPEA (312 mg, 2.4 mmol) was added followed by4-nitrophenyl chloroformate (161.3 mg, 0.8 mmol) and DMAP (98 mg, 0.8mmol). The resulting mixture was stirred for 15 minutes, then anadditional amount of 4-nitrophenyl chloroformate (161.3 mg, 0.8 mmol)was added. The reaction was stirred for 2 hours, then water (0.2 mL) wasadded to quench unreacted chloroformate. After stirring for 10 minutes,a solution of propargyl-PEG4-amine (259 mg, 1.12 mmol) in anhydrous DMF(0.5 mL) was added. The reaction was stirred for 1 hour and thenpurified directly by RPLC (100 g, 5 to 60% acetonitrile and water, using0.1% TFA as modifier). The collected fractions were lyophilized. Yield391.2 mg, 74.4%. Ion found by LCMS: [M+H]⁺=658.3, [M-Boc+H]⁺=558.3.

Step b

The step-a product (391.2 mg, 0.595 mmol) was dissolved in TFA (0.8 mL),and the solution was stirred at room temperature for 20 minutes. It wasthen directly purified by RPLC (100 g, 5 to 60% acetonitrile and water).Yield 323.2 mg, 81%. Ion found by LCMS: [M+H]⁺=558.3.

Step c

To a solution of the step-b product (323.2 mg, 0.482 mmol) in THF (2 mL)was added N,N′-Bis-Boc-1-guanylpyrazole (224.4 mg, 0.723 mmol) and PIPEA(260 mg, 2 mmol). The reaction mixture was stirred for 1 day and thenextracted with water (3 mL) and EtOAc/hexanes (1:1.8 mL). The organiclayer was dried over Na₂SO₄ and concentrated by rotary evaporation. Theresidue was re-dissolved in TFA (˜1 mL), and the solution was stirredovernight. It was then directly purified by RPLC (100 g, 0 to 30%acetonitrile and water, using 0.1% TFA as modifier). The collectedfractions were lyophilized. Yield 254.2 mg, 83.2%. Ions found by LCMS:[M+H]⁺=600.3, [M-Boc+H]⁺=300.6.

Step d

The step-c product (254.2 mg, 0.356 mmol) was dissolved in THF (1.5 mL),and the solution was cooled in an ice-water bath. CaCl₂ dihydrate (419mg, 2.85 mmol) was added, then 1.8 mL of KOH (112 mg, 2 mmol) in water(2 mL) was added in portions over 1 hour. After stirred for 3 morehours, the reaction mixture was acidified by Dowex 50W×8 hydrogen formand purified by RPLC (0 to 30% acetonitrile and water, using 0.1% TFA asmodifier). Yield 102 mg, 49.8%. Ions found by LCMS: [M+H]⁺=586.4,[(M+2H)/2]⁺=293.8.

Example 16. Synthesis of Conjugate 4

The title conjugate was prepared analogously to Conjugate 1 (Example 9)using Int-5 (Example 15). Maldi TOF analysis of the purified finalproduct gave an average mass of 63002 Da (DAR=3.4). Yield 49.315 mg, 49%yield. FIG. 12 shows a non-reducing SDS-PAGE of Conjugate 4.

Example 17. Synthesis of Int-6

Step a

A flame-dried reaction flask was flushed with nitrogen and charged with(1S,2S,3R,4R)-methyl3-((S)-1-acetamido-2-ethylbutyl)-4-(tert-butoxycarbonylamino)-2-hydroxycyclopentanecarboxylate (280.4 mg, 0.7 mmol) and anhydrous DCM (1 mL).After stirring to dissolve the starting material, DMAP (85.7 mg, 0.7mmol) was added to the solution, followed bybis(pentafluorophenyl)carbonate (295.6 mg, 0.75 mmol). The resultingmixture was stirred for 1 hour, then added into a solution of Linker-2(Example 5) (157.5 mg, 0.22 mmol) in anhydrous DMF (1 mL) and DIPEA (130mg, 1 mmol). The reaction was stirred overnight and purified by RPLC (50g, 5 to 90% acetonitrile and water). The collected fractions werelyophilized. Yield 134.1 mg, 40.7%. Ion found by LCMS:[(M+2H)/2]⁺=748.6.

Step b

The step-a product (134.1 mg, 0.0896 mmol) was dissolved in TFA (0.5mL). The solution was stirred for 20 minutes, then directly purified byRPLC (50 g, 5 to 60% acetonitrile and water). Yield 108.4 mg, 93.4%.Ions found by LCMS: [(M+2H)/2]⁺=648.3, [(M+3H)/3]⁺=432.8.

Step c

To a solution of the step-b product (108.4 mg, 0.0837 mmol) in anhydrousTHF (1 mL) was added N,N′-bis-boc-1-guanylpyrazole (81.3 mg, 0.285 mmol)and DIEPA (65 mg, 0.5 mmol). The reaction was stirred at roomtemperature for 2.5 days, then directly purified by RPLC (100 g, 40 to75% acetonitrile and water). Yield 73.6 mg, 49.4%. Ion found by LCMS:[(M+3H)/3]⁺=594.2.

Step d

The step-c product (73.6 mg, 0.041 mmol) was dissolved in TFA (0.5 mL).The solution was stirred for 20 minutes, then directly purified by RPLC(50 g, 5 to 60% acetonitrile and water). Yield 62.1 mg, 94.1%. Ionsfound by LCMS: [(M+2H)/2]⁺=690.4, [(M+3H)/3]⁺=460.8.

Step e

The step-d product (62.1 mg, 0.0386 mmol) in THF (3 mL) was cooled in anice-water bath and a 45% w/w solution of KOH (0.2 mL) was added inportions over 1 hour. The reaction was stirred for 2 more hours, thenacidified with 4N HCl solution in dioxane (0.8 mL) and extracted withhexanes (10 mL) and water (1.5 mL). The aqueous layer was purified byHPLC (0 to 20% acetonitrile and water, using 0.1% TFA as modifier).Yield 36.2 mg, 59.4%. Ions found by LCMS: [(M+2H)/2]⁺=676.5,[(M+3H)/3]⁺=451.4.

Example 18. Synthesis of Conjugate 5

The title conjugate was prepared analogously to Conjugate 1 (Example 9)using Int-6 (Example 17). Maldi TOF analysis of the purified finalproduct gave an average mass of 63561 Da (DAR=3.3). Yield 43.4 mg, 43%yield. FIG. 13 shows a non-reducing SDS-PAGE of Conjugate 5.

Example 19. Synthesis of Int-7

Step a

To a solution of propargyl-PEG4-acid (609 mg, 2.34 mmol) andNH-bis(PEG₁-azide) (500 mg, 2.055 mmol) in anhydrous DMF (2 mL) wasadded HATU (889.7 mg, 2.34 mmol) in portions over 5 minutes. Afterstirring to dissolve all the coupling reagent, DIPEA (390 mg, 3 mmol)was added and stirring continued for 1 hour. It was then purifieddirectly by RPLC (100 g, 5 to 40% acetonitrile and water). Yield 918 mg,92%. Ion found by LCMS: [M+H]⁺=486.2.

Step b

The step-a product (918 mg, 1.89 mmol) was dissolved in THF, and thesolution was cooled to 13° C. Triphenylphosphine (1.141 g, 4.35 mmol)was added in portions over 10 minutes. The resulting mixture was stirredat 13° C. to room temperature for 2 hours. A solution of LiOHmonohydrate (42 mg, 1 mmol) in water (2 mL) and MeOH (1 mL) was added.After stirring was continued for 20 hours, the reaction was purified byRPLC (100 g, 0 to 30% acetonitrile and water, using 0.1% TFA asmodifier). Yield 825 mg, 65.9%. Ion found by LCMS: [M+H]⁺=434.4.

Step c

A flame-dried reaction flask was flushed with nitrogen and charged withZanamivir intermediate (Example 2) (865 mg, 1.51 mmol) and anhydrous DCM(5 mL). After stirring to dissolve the starting material, the solutionwas cooled in an ice-water bath and 4-nitrophenyl chloroformate (365.3mg, 1.81 mmol) was added followed by DMAP (152.2 mg, 0.755 mmol). Theice-water bath was removed, and the mixture was stirred for 3 hours. Anadditional amount of 4-nitrophenyl chloroformate (304.4 mg, 1.51 mmol)was added, and stirring was continued for 1 hour. The reaction was thenquenched with water (1 mL). After vigorously stirred for 1 hour, thereaction mixture was extracted with water (20 mL×2) and DCM (20 mL). Theorganic layer was stirred overnight, dried over Na₂SO₄, and concentratedby rotary evaporation. The material was carried on to the subsequentstep without further purification. Ion found by LCMS: [M+H]⁺=738.2.

Step d

A solution of a mixture of the step-b product (429.7 mg, 0.65 mmol) andDIPEA (260 mg, 2 mmol) in anhydrous THF (1 mL) was added dropwise to thestep-c product. The resulting mixture was stirred for 2 hours, thendirectly purified by RPLC (100 g, 10 to 65% acetonitrile and water).Acetonitrile in the collected fractions was removed by rotaryevaporation at room temperature. The heterogeneous aqueous layer wasextracted with EtOAc (200 mL), then back-extracted with EtOAc (50 mL).The combined organic layers were dried over Na₂SO₄ and concentrated byrotary evaporation to dryness. Yield 442 mg, 41.7%. Ions found by LCMS:[(M+2H)/2]⁺=815.8, [(M-Boc+2H)/2]⁺=765.8, [(M-2Boc+2H)/2]⁺=716.

Step e

The step-d product (441 mg, 0.271 mmol) was dissolved in TFA (1 mL). Thesolution was stirred for 20 minutes, then directly purified by HPLC (5to 20% acetonitrile and water, using 0.1% TFA as modifier). Yield 308mg, 77.9%. Ions found by LCMS: [(M+2H)/2]⁺=615.8, [(M+3H)/3]⁺=411.

Step f

The step-e product (308 mg, 0.211 mmol) was dissolved in MeOH (1 mL) andwater (0.5 mL), and the solution was cooled in an ice-water bath. Asolution of LiOH monohydrate (42 mg, 1 mmol) in water (1 mL) was addedin portions over 1 hour. The reaction was stirred overnight, acidifiedby 4 N HCl solution in dioxane (0.25 mL), and purified by RPLC (0 to 20%acetonitrile and water, using 0.1% TFA as modifier). Yield 198 mg, 64%.Ions found by LCMS: [(M+2H)/2]⁺=575.8, [(M+3H)/3]⁺=384.2.

Example 20. Synthesis of Conjugate 6

The title conjugate is prepared analogously to Conjugate 1 (Example 9)using Int-7 (Example 19). Maldi TOF analysis of the purified finalproduct gave an average mass of 62854. Da (DAR=3.1). Yield 175.4 mg, 50%yield. FIG. 14 shows a non-reducing SDS-PAGE of Conjugate 6.

Example 21. Synthesis of Int-8

Step a

Methyl5-acetoamido-7,8,9-O-triacetyl-2,6-anhydro-4-azido-3,4,5-trideoxy-D-glycero-D-galacto-non-2-enonate(1.8 g, 4.0 mmol) was dissolved into 40 mL methanol and then treatedwith 400 mg of 5% Pd/C and 1.1 g Boc anhydride (5.0 mmol), then thereaction mixture was stirred at room temperature under a hydrogenatmosphere for 1 hour. The palladium-charcoal was removed by filtration.The filtrate was concentrated and used in next step withoutpurification.

Step b

The product from the previous step was dissolved into 20 mL dry methanoland then treated with 2 mL sodium methoxide in methanol (0.5 M) dropwisewith cooling in an ice-water bath. After 2 hours, the progress ofreaction was determined by LCMS. The reaction was quenched with 1N HClto pH 5-6. The resulting solution was concentrated and purified byreversed phase liquid chromatography (RPLC) using an Isco COMBIFLASH®liquid chromatograph eluted with 5% to 100% acetonitrile and water,using 0.1% TFA as the modifier, Ion(s) found by LCMS: (M+H)⁺=405

Step c

Methyl5-acetamido-2,6-anhydro-4-[(tert-butoxycarbonyl)amino]-3,4,5-trideoxy-D-erythro-non-2-enonate(0.4 g, 1 mmol) was dissolved in 10 mL of acetone, 4 mL of2,2-dimethoxypropane and 20 mg of p-toluenesulfonic acid hydrate (0.1mmol). The resulting solution was stirred at room temperature overnight,then quenched with 1 mL saturated NaHCO₃. The mixture was concentratedand purified by reversed phase liquid chromatography (RPLC) using anIsco COMBIFLASH® liquid chromatograph eluted with 5% to 100%acetonitrile and water without modifier. Ion(s) found by LCMS:(M+H)⁺=445.

Step d

Sodium hydride (40 mg, 60% in oil, 1.0 mmol) was added into methyl5-acetamido-2,6-anhydro-4-[(tert-butoxycarbonyl)amino]-3,4,5-trideoxy-8,9-O-(1-methylethylidene)-D-erythro-non-2-enonate(0.25 g, 0.50 mmol) in 5 mL dry THF with cooling from an ice-water bath.The resulting solution was stirred for 0.5 hour, then benzylbromoacetate (0.23 g, 1.0 mmol) was added. The resulting solution wasstirred for 2 hours and quenched with 5 mL 10% ammonium chloride inwater. Then the solution was diluted with 50 mL ethyl acetate. Theorganic layer was separated and dried with sodium sulfate. The driedorganic solution was concentrated and purified by reversed phase liquidchromatography (RPLC) using an Isco COMBIFLASH® liquid chromatographeluted with 5% to 100% acetonitrile and water without modifier. Ion(s)found by LCMS: (M+H)⁺=593.

Example 22. Synthesis of Int-9

Step a

Methyl5-acetoamido-7,8,9-O-triacetyl-2,6-anhydro-4-azido-3,4,5-trideoxy-D-glycero-D-galacto-non-2-enonate(10 g, 22 mmol) was dissolved into 100 ml methanol and then heated to60° C. with an oil bath, the SnCl₂ (5.7 g, 20 mmol) was added to thesolution in 3 portions (caution, gas evolves). The reaction mixture wasstirred for 10 min, at which time the reaction was complete by HPLC. Thereaction solution was slowly added to a solution of 50 ml Sat NaHCO₃ and50 g celite with vigorous stirring. The resulting slurry was filtered.The filtrate was treated with Boc₂O (6.6 g, 30 mmol, 1.5 equiv). After 2hour at room temperature, the solution was concentrated to remove mostof the methanol, dissolved in 200 ml DCM, and extracted twice with 100ml DCM. The combined extracts were dried with sodium sulfate, filteredand used for next step without further purification. Crude yield 12 g,100%. Ion(s) found by LCMS: M+H=531.

Step b

The material from the previous step was dissolved into 60 ml drymethanol, then treated with 10 ml sodium methoxide in methanol (0.5M)while cooling with an ice-water bath. Progress of reaction was monitoredby LCMS which was complete after 2 h. The reaction was quenched with 1NHCl to a pH of 5-6. The resulting solution was concentrated and purifiedby reverse phase liquid chromatography (RPLC) using an Isco COMBIFLASH®liquid chromatograph eluted with 0% to 30% acetonitrile and water, using0.1% TFA as the modifier. Yield of the products 7.2 g, 80%. Ion(s) foundby LCMS: M+H=405.

Step c

A solution of the product from the previous step (3.5 g, 8.5 mmol), CDI(2.8 g, 2 equiv), trimethylamine (4.2 ml, 30 mmol) and DMAP (240 mg, 2mmol) were heated in acetonitrile (50 ml) overnight, then concentratedand purified by reverse phase liquid chromatography (RPLC) using an IscoCOMBIFLASH® liquid chromatograph eluted with 0% to 30% acetonitrile andwater without modifier. Yield of desired product 2.3 g, 60%. Ion(s)found by LCMS: M+H=431.

Step d

Sodium hydride (400 mg, 60% in oil, 10 mmol) was added to the productfrom the previous step (1.45 g, 3.3 mmol) in 50 ml dry THF(moisture-sensitive reaction) under the ice-water bath. The resultedsolution was stirred for 0.5 hour, then tertbutyl bromo-acetate (2 g, 10mmol) was added to the above solution, the resulted solution was heatedupto 60° C. for overnight and quenched with acetic acid. The resultingsolution was concentrated and purified by reverse phase liquidchromatography (RPLC) using an Isco COMBIFLASH® liquid chromatographeluted with 0% to 50% acetonitrile and water with TFA as modifier. Yieldof 1 g, 57%. Ion(s) found by LCMS: M+H=593.

Step e

The product from the previous step (1.2 g, 2.2 mmol) was stirred with 10ml TFA at room temperature for overnight, and the progress ofdeprotection was monitored by LCMS. The resulted solution wasconcentrated and used for next step without purification. The residuewas re-dissolved into 20 ml THF, then N,N′-bis-boc-1-guanylpyrazole (1g, 3.3 mmol), 4-dimethylaminopyridine (120 mg, 1 mmol) and triethylamine(0.7 ml, 5 mmol) were added to the solution, and the resulting solutionwas heated to 60° C. for 2 hours. The resulting solution wasconcentrated and purified by reverse phase liquid chromatography (RPLC)using an Isco COMBIFLASH® liquid chromatograph eluted with 0% to 50%acetonitrile and water with no modifier. Yield of 700 mg, 84%. Ion(s)found by LCMS: M+H=631.

Step f

To a solution of linker-3 (prepared as described in Example 19) (73 mg,0.14 mmol) and the product of the previous step (200 mg, 0.32 mmol, 2.2equi) in DMF (30 ml) was added EDC (100 mg, 0.5 mmol), HOAt (65 mg, 3mmol), and DIEA (0.14 ml, 1 mmol) at room temperature. The solution wasstirred overnight. The resulting solution was concentrated and purifiedby and purified by reverse phase liquid chromatography (RPLC) using anIsco COMBIFLASH® liquid chromatograph eluted with 0% to 50% acetonitrileand water with no modifier. Yield of 120 mg, 52%. Ion(s) found by LCMS:M/2+H=830.

Step g

Lithium hydroxide (24 mg, 1 mmol) in 2 ml H2O was added into thesolution of the product from the previous step (120 mg, 0.07 mmol) in 2ml THF and 1 ml MeOH, LCMS monitored the progress of the reaction. Afterthe completion, the solution was added AMBERLITE® IRN-77, ion exchangeresin to adjust to pH1, then the resulting solution was filtered and thefiltrate was concentrated and used for next step without purification.The resulting compound was treated with 2 ml TFA at room temperature,the solution was stirred for overnight at 40° C., then concentrated andpurified by HPLC eluted with 0% to 20% acetonitrile and water, using TFAas the modifier. Yield 60 mg, 74% yield, Ion(s) found by LCMS:[M/2]+1=589.8.

Example 23. Neuraminidase Inhibition Assay

A neuraminidase inhibition assay using2′-(4-Methylumbelliferyl)-alpha-D-N-aceylneuraminic acid (MUNANA)substrate was performed as described below. Briefly, 50 μL of purified,recombinant influenza virus neuraminidase (0.1 ng/μL, 50 mM Tris, 5 mMCaCl2, 200 mM NaCl, pH 7.5) was mixed with 50 μL of inhibitor andincubated for 30 min at room temperature. At least 5 concentrations ofeach inhibitor at an appropriate range were used for each repeat.Following incubation, 50 μL of 400 μM MUNANA in 50 mM Tris, 5 mM CaCl2,200 mM NaCl, (pH 7.5) was added to the solution to start the reactionusing a 12-tip pipette (Eppendorf). A positive and a negative controlwere included in each 12-well lane. After starting the reaction for eachlane on the plate, the reaction mixture was immediately loaded on aSpectraMax M5 (Molecular Devices) where fluorescence was quantified overthe course of 25 min at an excitation wavelength of 365 nm and anemission wavelength of 445 nm. Single time points were chosen where thepositive control produced a fluorescence signal of approximately 1,000.All assays were done in triplicates and IC50 values for each inhibitorwere calculated with sigmoidal fitting of the log[inhibitor] vs.inhibition percentage using GraphPad Prism. FIG. 15 shows the plot ofthe kinetic data as RFU/min over a linear range for Conjugate 1 andInt-2 showing the greater efficacy of Conjugate 1 as a neuraminidaseinhibitor. FIG. 16 and FIG. 17 show the assay results for Conjugates 1-6against rH1N1 Neuraminidase and rH3N2 Neuraminidase, respectively (Table2).

TABLE 2 IC50 values for conjugates against H1N1 and H3N2 NeuraminidaseConjugate Number H1N1 IC50 (nM) H3N2 IC50 (nM) Conjugate 1 3.9 32.5Conjugate 2 3.7 17.6 Conjugate 3 3.6 38 Conjugate 4 1.9 17.2 Conjugate 51.1 14.3 Conjugate 6 4.1 29.8

Example 24: Cytotoxicity Assay

Conjugates 1, 2, 3, and 6 were tested for cytotoxicity. Ten two-foldserial dilutions of each conjugate starting at 10 μM were prepared intriplicate for inoculation with MDCK cells in 96-well culture plates.The cell by way ofbility was determined four days post treatment usingCellTiter-GLO kit. 50% of cytotoxicity concentration (CC₅₀) wascalculated using XLfit dose response model (Table 3). For all compoundstested, cytotoxicity was not observed up to the 10 μM, with theexception of conjugate 3. In the case of Conjugate 3, The EC₅₀ in thecytopathic effect (CPE) assay (see Example 23) is 725-fold below theCC₅₀ in this assay.

TABLE 3 Cytotoxicity test Conjugate Number CC50 (μM) Conjugate 1 >10Conjugate 2 >10 Conjugate 3 7.98 Conjugate 6 >10 PBS >10 Zanamavir >10

Example 25. Cytopathic Effect Assay CPE-Based Microneutralization Assay#1

To measure the ability of neuraminidase conjugates to protect mammaliancells from infection and destruction by influenza virus, cytopathiceffect (CPE)-based microneutralization assays were conducted. Briefly,twenty two-fold serial dilutions of each conjugate starting at 0.25 μMwere prepared in duplicate for one-hour inoculation with MDCK cellsseeded in 96-well plates. INFV CA/09 virus was added to cells at amultiplicity of infection (MOI) 0.001 for one-hour incubation. On dayfour post incubation, cells were stained with crystal violet and opticaldensity was read for calculation of 50 percent effective concentration(EC₅₀) of each TA using XLfit dose response model. Zanamivir was used asa comparator and positive control, human Fc was included as a negativecontrol. Conjugates 1, 2, 3, and 6 all demonstrated superior performanceto the zanamivir control, demonstrating EC50s 42- to 227-fold lower thanzanamivir (Table 4).

TABLE 4 CPE-based microneutralization assay #1 Conjugate Number EC50(nM) 1 2.0574 2 4.1487 3 11.007 6 5.2167 PBS N/C Human Fc N/CZanamavir^(†) 468.7 N/C: Not calculatable ^(†)Starting concentration10000 nM

CPE-Based Microneutralization Assay #2

An additional CPE-based microneutralization assay was run to furtherevaluate the in vitro activity of Conjugate 6 and Conjugate 7. Briefly,test articles were prepared at a starting concentration of 160 nM, and atotal often, 2-fold dilutions were made. The test article dilutions werethen pre-mixed with Influenza A virus (INFV CA/09) at a multiplicity ofinfection of 0.001 relative to the monolayer. After one 10 hour the testarticle+viral mix was added to Madin-Darby Canine Kidney (MDCK) cellsgrown to 70-80% confluence under standard conditions in a 96-well plate.The zanamivir control was treated the same, except the startingconcentration was 9600 nM since it was expected to be less potent thanthe Conjugates being tested. After four days of incubation the monolayerwas stained with crystal violet and optical density (reflective ofmonolayer health) was determined to calculate the 50% effectiveconcentration (EC50) using the XLfit dose response model (idbs;https://www.idbs.com).

As shown in Table 5, zanamivir at a concentration of 496 nM reduced thevirus mediated cytopathic effects by half. In contrast, Conjugate 6 wasapproximately 100× more active with an EC50 of only 4.64 nM. Conjugate7, further improved activity by approximately 15-fold, reducing the EC50to the sub-nM level.

TABLE 5 CPE-based microneutralization assay #2 Conjugate Number EC50(nM) 6 4.64 7 0.31 Zanamivir 496

Example 26. Cell by Way Ofbility Assay

A549 cells were seeded in 96-well plates one day prior to compoundtreatment. Cells were treated with either Conjugate 3 (FIG. 18A),Conjugate 4 (FIG. 18B), or Conjugate 6 (FIG. 18C) at concentrations 1μM-10 nM for 24 hours. Cell by way ofbility was then measured using CellTiter Glow (Promega). Results represent average and SD of threebiological replicates. No effects were observed on cell by way ofbilityfor any of the conjugates at any concentrations at any of theconcentrations used in the viral growth assay (Example 27).

Example 27. Viral Growth Assay

To measure the ability of neuraminidase conjugates to inhibit the growthof pathogenic influenza viral strains of interest in human epithelialcells, viral plaque reduction assays were conducted. Briefly, A549 cellswere seeded in 24-well plates one day prior to compound treatment. Cellswere treated with compounds at concentrations 1 μM-10 nM for 2 hours andinfected with indicated viral strains at MOI 0.01 for one-hourincubation. Virus was removed, cells were washed and compounds werere-applied at concentrations 1 μM-10 nM. Supernatants were collected atindicated time points and titrated using plaque assay method in MDCKcells. Results represent average and SD of three biological replicates.Oseltamivir was used as a comparator and a positive control. Conjugate 3(FIGS. 19A-19E), Conjugate 4 (FIGS. 20A-E), or Conjugate 6 (FIGS.21A-21E and FIGS. 22A-22E) all demonstrated superior performance to theoseltamivir control, showing similar or (usually) superior plaquereduction to oseltamivir at 100-fold lower concentrations. The effectsof the compounds (to evaluate test articles for potential cytotoxicity)on the A549 cells was evaluated for each test article using a cell byway ofbility assay (Example 26). Briefly, A549 cells were seeded in96-well plates one day prior to compound treatment. Cells were treatedwith compounds at concentrations 1 μM-10 nM for 24 hours. Cell by wayofbility was then measured using Cell Titer Glow (Promega). Resultsrepresent average and SD of three biological replicates.

Example 28. Mouse Serum Half-Life

Pharmacokinetic (PK) studies used female CD-1 mice (Charles RiverLaboratories) between 20 and 22 grams. Mice were injected IV by way ofthe tail vein, with 50 mg/kg of test article (10 ml/kg dose volume).Animals were housed under standard IACUC approved housing conditions. Atappropriate times animals were non-terminally bled (retro-orbital,cheek, or by tail vein) with blood collected in EDTA tubes to preventcoagulation. Collected blood was centrifuged (2,000×g, for 10 minutes)and plasma withdrawn for analysis of test article concentrations overtime.

The plasma concentrations for Conjugate 6 or hIgG1 Fc at each time pointwere measured by sandwich ELISA as follows: Conjugate 6 molecules werecaptured either on Neuraminidase coated plates or anti-hIgG1 antibodycoated plates and then detected using an HRP-conjugated anti-humanIgG-Fc antibody. hIgG1 was captured using anti-hIgG1 Fc antibody.Protein concentration was calculated in GraphPad Prism using 4PLnon-linear regression of Conjugate 6 (or hIgG1 Fc) standard curves. Amore detailed method description is provided below.

Qiagen Ni-NTA HisSorb plates (Cat No. 35061, Qiagen) were coated witheither Neuraminidase from A/California/04/2009 (H1N1) (11058-VNAHC, SinoBio) or anti-IgG1 Fc antibody in 1×KPL coating buffer (5150-0041,SeraCare). In the cases where the anti-IgG1 Fc antibody was used tocapture test article, capture and detection anti-IgG1 Fc antibodies wereselected that bind different epitopes. Plates were incubated at roomtemperature for 1 hour. Serial dilutions of the plasma samples wereplated and incubated at room temperature for 2 hrs (sample diluent: 0.5%nonfat dry milk+3% Goat serum in PBS 0.05% Tween20; naïve mouse plasmafinal concentration of 1:900). Conjugate 6 or hIgG1 Fc standard curvesranging from 500-0.230 ng/mL, in duplicate were run on each plate.

Following the 2 hr incubation, plates were washed 5× in 300 uL PBS with0.05% Tween20. Conjugate 6 bound to neuraminidase on the plates (oranti-hIgG1 Fc antibody) was then probed with an HRP conjugatedanti-human IgG Fc F(ab′)2 (Jackson 709-036-098) diluted 1:2000 in samplediluent for 1 hour at room temp. Plates were then washed 8× in 300 uLPBS with 0.05% Tween20 and developed with TMB substrate for 7 minutes.The reaction was stopped with 1N H2S04. Absorbance was read at 450 nm. Asimilar protocol was used for hIgG1 Fc, where only anti-hIgG1 Fcantibody was used for capture. The quantities of Conjugate 6 measured atdifferent timepoints using either neuraminidase or anti-hIgG1 Fcantibody capture were similar within experimental error, suggesting thatthe intact conjugate is stable in vivo.

Total Conjugate 6 (or hIgG1 Fc) in test samples was interpolated usingin Graphpad Prism Version 6 following nonlinear regression analysis(Sigmoidal, 4PL analysis) of the Conjugate 6 (or hIgG1 Fc) standardcurves. PK parameters were calculated using WinNonlin software. Thecurves comparing Conjugate 6 and hIgG1 Fc are shown in FIG. 23 and asummary of key PK parameters is provided in Table 6. Unexpectedly,plasma exposures and terminal half-life are significantly better forConjugate 6 than for wild-type hIgG1 Fc. The AUCs over 8-days are 3×higher for Conjugate 6 than for hIgG1 Fc, and the terminal half-life forConjugate 6 is 214 hours, versus 52 hours for human IgG1 Fc.

TABLE 6 Mouse PK of Conjugate 6 compared to hIgG1 Fc Half-life (hrs)AUClast (hr * mg/mL) Conjugate 6 214 33100 hIgG1 51.8 10600

Example 29. Efficacy of Conjugate 6 in a Lethal Mouse Influenza Model:Study #1

Conjugate 6 was evaluated against a lethal INFV A H1N1 influenzainfection in female BALB/c mice. The experiment comprised 7 groups of 5mice. At day 0, all mice were challenged with 1×LD90 H1N1 A/Texas/36/91.Groups 1-6 received treatment IV, 4 hours before challenge (Table 7).Human IgG1 (Fc alone) was included as an additional negative control.Group 7 received Oseltamivir phosphate by way of oral delivery, starting8 hours post infection twice daily for 5 days. All mice were monitoredfor weight loss (FIG. 24, Table 8) and survival (FIG. 25, Table 9) for15 days after challenge. Mice treated with Conjugate 6 showed 100%survival with single doses in all the concentrations tested, comparedwith 20% and 0% survival in the vehicle control and hIgG1 controlgroups, respectively. The results were statistically significant whencompared to the vehicle group (p=0.0135) despite the small group size(n=5). When compared to the Oseltamivir phosphate group, Conjugate 6demonstrated similar efficacy at a 500× lower cumulative dose (inmg/kg). Mice at all Conjugate 6 doses maintained their weight throughthe entire course of the experiment, superior to the Oseltamivir controlgroup.

TABLE 7 Study design Group Challenge Dose Treatment n = 5 Day 0 Compound(mg/kg) Route/Schedule 1 Influenza A virus, Vehicle (PBS) N/A IV, q.d. 4hours pre-challenge 2 H1N1 strain Fc alone 50 3 A/Texas/36/91 by wayConjugate 6 50 4 of IN route. Conjugate 6 10 5 Conjugate 6 2 6 Conjugate6 0.4 7 Oseltamivir (Tamiflu ™) 20 PO, b.i.d. 8 hours after challengefor 5 days

TABLE 8 Daily Weight Average Daily average weight (The mice number isadded only for Group 1 & 2) Oseltamivir Days post Fc alone Conjugate 6phosphate infection Vehicle (PBS) 50 mg/kg 50 mg/kg 10 mg/kg 2 mg/kg 0.4mg/kg 20 mg/kg 0 17.9 (5) 17.9 (5) 18.7 19.3 18.5 18.8 18.8 1 18.0 (5)18.0 (5) 18.8 19.36 18.6 18.8 18.9 2 18.5 (5) 18.8 (5) 19.2 19.6 19.418.8 19.2 3 17.7 (5) 17.7 (5) 18.9 19.5 19.1 18.9 19.1 4 17.2 (5) 17.0(5) 19.5 19.9 19.3 18.9 18.9 5 16.3 (5) 15.8 (5) 19.2 19.8 19.5 18.718.8 6 15.6 (4) 15.0 (5) 19.1 19.9 19.5 18.7 18.4 7 15.5 (2) 14.0 (2)18.8 19.6 19.2 18.9 17.3 8 17.2 (1) 19.1 20.0 19.7 19.2 17.1 9 20.1 (1)18.7 19.3 19.1 18.8 17.8 10 20.0 (1) 19.1 20.1 19.5 19.3 18.9 11 20.4(1) 19.0 19.8 19.2 19.2 18.7 12 20.6 (1) 19.6 20.3 19.8 19.3 19.2 1320.3 (1) 19.2 20.4 19.7 19.7 19.3 14 20.7 (1) 19.5 20.6 19.9 19.8 19.6

TABLE 9 Mouse survival Mean Significance Compound Dosage Survival %Survival to vehicle (p) Vehicle (PBS) N/A 7 20 N/A Fc alone 50 mg/kg 7 00.8335 Conjugate 6 50 mg/kg 15 100 0.0135 Conjugate 6 10 mg/kg 15 1000.0135 Conjugate 6  2 mg/kg 15 100 0.0135 Conjugate 6 0.4 mg/kg  15 1000.0135 Oseltamivir 20 mg/kg B.I.D 15 100 0.0135 phosphate

Example 30. Efficacy of Conjugate 6 in a Lethal Mouse Influenza Model:Study #2

Conjugate 6 was evaluated against a lethal INFV A H3N2 influenzainfection in female BALB/c mice. The experiment comprised 11 groups of 5mice. At day 0, all mice were challenged with 1×LD90 H3N2 A/HongKong/1/68. Groups 1-10 received treatment IV, 4 hours before challenge(Table 10). Human IgG1 (Fc alone) was included as an additional negativecontrol. Group 11 received Oseltamivir phosphate by way of oraldelivery, starting 8 hours post infection twice daily for 5 days. Allmice were monitored for weight loss (FIG. 26) and survival (FIG. 27,Table 11) for 15 days after challenge. Mice treated with Conjugate 6showed 100% survival with single doses in down to 0.4 mg/kg, and 80%survival with a single dose of 0.2 mg/kg compared with 0% survival inthe vehicle control and hIgG1 control groups, respectively. 80% of themice survived in the oseltamivir control group. The results werestatistically significant when compared to the vehicle group (p=0.0128)despite the small group size (n=5). When compared to the Oseltamivirphosphate group, Conjugate 6 demonstrated similar efficacy at a 1000×lower cumulative dose (in mg/kg). Mice at all Conjugate 6 dosesmaintained down to 0.4 mg/kg their weight within 5%, superior to theOseltamivir control group.

TABLE 10 Study design Group Challenge Dose Treatment n = 5 Day 0 TestArticle (mg/kg) Route/Schedule Readout/Endpoint  1 H3N2 Vehicle N/A IV,q.d. 4 hours Daily weight and health A/Hong (PBS) pre-challenge scoremonitoring for 15  2 Kong/1/68 Fc alone 50 days after challenge  3 byway of Conjugate 6 50 % Survival  4 IN route 2  5 0.4  6 0.2  7 0.1  80.05  9 0.025 10 0.0125 11 Oseltamivir 20 PO, b.i.d. 8 hours (Tamiflu ™)after challenge for 5 days

TABLE 11 Mouse survival Mean Significance Survival to vehicle CompoundDosage (days) % Survival (p-value) Vehicle (PBS) N/A 9 0 N/A Fc control50 mg/kg 8 0 0.2498 Conjugate 6 50 mg/kg 15 100 0.002 2 mg/kg 15 1000.002 0.4 mg/kg 15 100 0.002 0.2 mg/kg 15 80 0.0128 0.1 mg/kg 9 200.8264 0.05 mg/kg 9 20 0.5769 0.025 mg/kg 8 20 >0.9999 0.0125 mg/kg 11 00.4703 Oseltamivir 20 mg/kg 15 80 0.0052 phosphate (B.I.D for 5 days)

Example 31. Synthesis of Int-10

Step a

Methyl5-acetamido-7,8,9-O-triacetyl-2,6-anhydro-4-azido-3,4,5-trideoxy-D-glycero-D-galacto-non-2-enonate(SM-1, 10 g, 22 mmol) was dissolved into 100 ml methanol and then heatedto 60° C. with an oil bath, the SnCl₂ (5.7 g, 20 mmol) was added to thesolution in 3 portions (caution, gas evolves). The reaction mixture wasstirred for 10 min, at which time the reaction was complete by HPLC. Thereaction solution was slowly added to a solution of 50 ml Sat NaHCO₃ and50 g celite with vigorous stirring. The resulting slurry was filtered.The filtrate was treated with Boc₂O (6.6 g, 30 mmol, 1.5 equiv). After 2hour at room temperature, the solution was concentrated to remove mostof the methanol, dissolved in 200 ml DCM, and extracted twice with 100ml DCM. The combined extracts were dried with sodium sulfate, filteredand used for next step without further purification. Crude yield 12 g,100%. Ion(s) found by LCMS: M+H=531.

Step b

The material from the previous step was dissolved into 60 ml drymethanol, then treated with 10 ml sodium methoxide in methanol (0.5M)while cooling with an ice-water bath. Progress of reaction was monitoredby LCMS which was complete after 2 h. The reaction was quenched with 1NHCl to a pH of 5-6. The resulting solution was concentrated and purifiedby reverse phase liquid chromatography (RPLC) using an Isco COMBIFLASH®liquid chromatograph eluted with 0% to 30% acetonitrile and water, using0.1% TFA as the modifier. Yield of the products 7.2 g, 80%. Ion(s) foundby LCMS: M+H=405.

Step c

A solution of intermediate from the previous step (3.5 g, 8.5 mmol), CDI(2.8 g, 2 equiv), trimethylamine (4.2 ml, 30 mmol) and DMAP (240 mg, 2mmol) were heated at 60° C. in DMF (50 ml) overnight, then concentratedand purified by reverse phase liquid chromatography (RPLC) using an IscoCOMBIFLASH® liquid chromatograph eluted with 0% to 30% acetonitrile andresulting without modifier. Yield of desired product 2.3 g, 60%. Ion(s)found by LCMS: M+H=431.

Step d

Sodium hydride (400 mg, 60% in oil, 10 mmol) was added to Int-4 (1.45 g,3.3 mmol) in 50 ml dry THF (moisture-sensitive reaction) under theice-water bath. The resulting solution was stirred for 0.5 hour, thentert-butyl bromo acetate (2 g, 10 mmol) was added to the above solution.The resulting solution was heated to 60° C. overnight and quenched withacetic acid, concentrated and purified by reverse phase liquidchromatography (RPLC) using an Isco COMBIFLASH® liquid chromatographeluted with 0% to 50% acetonitrile and water with TFA as modifier. Yieldof 1 g, 57%. Ion(s) found by LCMS: M+H=593. (The reaction is prettyclean by HPLC, but low isolated yield)

Step f

Intermediate from the previous step (1.2 g, 2.2 mmol) was stirred with10 ml TFA at room temperature overnight. The progress of deprotectionwas monitored by LCMS. The resulting solution was concentrated and usedin the next step without purification. The residue from the previousreaction was dissolved into 20 ml THF, thenN,N′-bis-boc-1-guanylpyrazole (1 g, 3.3 mmol), 4-dimethylaminopyridine(120 mg, 1 mmol) and triethyl amine (0.7 ml, 5 mmol) were added to thesolution, and the resulting solution was heated to 60° C. for 2 hours.The resulting solution was concentrated and purified by reverse phaseliquid chromatography (RPLC) using an Isco COMBIFLASH® liquidchromatograph eluted with 0% to 50% acetonitrile and water with nomodifier. Yield of 700 mg, 84%. Ion(s) found by LCMS: M+H=631.

Step g

To a solution of linker-3 (prepared as described in Example 19) (73 mg,0.14 mmol) and intermediate from the previous step. (200 mg, 0.32 mmol,2.2 equi) in DMF (30 ml) was added EDC (100 mg, 0.5 mmol), HOAt (65 mg,3 mmol), and DIEA (0.14 ml, 1 mmol) at room temperature. The solutionwas stirred overnight. The resulting solution was concentrated andpurified by and purified by reverse phase liquid chromatography (RPLC)using an Isco COMBIFLASH® liquid chromatograph eluted with 0% 15 to 50%acetonitrile and water with no modifier. Yield of 120 mg, 52%. Ion(s)found by LCMS: M/2+H=830.

Step h

Lithium hydroxide (24 mg, 1 mmol) in 2 ml water was added to a solutionof intermediate from the previous step (120 mg, 0.07 mmol) in 2 ml THFand 1 ml MeOH. After the reaction is complete by LCMS, the solution wasquenched with AMBERLITE® IRN-77, ion exchange resin to adjust to pH=1,then the solution was filtered and the filtrate was concentrated and inthe next step without further purification. Intermediate from theprevious step was treated in 2 ml TFA at room temperature overnight at40° C. The crude reaction was concentrated and purified by HPLC with 0%to 20% acetonitrile and water, using TFA as the modifier. Yield 60 mg,74% yield, Ion(s) found by LCMS: [M/2]+1=589.8.

Example 32. Synthesis of Conjugate 7

A solution of Fc-PEG4-azide (each Fc domain monomer having the sequenceof SEQ ID NO: 38) in PBS×1 buffer solution (100 mg, 1.28 μmol, 6.1 mL,MW=57260 Da, DAR=3.6) were added to Int-10 (prepared as described inExample 31) (TFA salt, 44 mg, 0.031 mmol) and freshly prepared pH 7.4PBS solutions of CuSO₄ (0.7 mL of 50.0 mM, 20 eq),tris(3-hydroxypropyltriazolylmethyl)-amine (THPTA, 0.7 mL of 50.0 mM, 20eq), and sodium ascorbate (1.05 mL of 50.0 mM, 30 eq). The resultinghomogeneous solution was agitated by rocker table for 12 h. The crudesolutions were diluted with pH 7.4 PBS to a final concentration of 1mg/mL, and ultrafiltered (10,000 MWCO) to a volume of 1 mL, two times.The crude mixtures were then diluted 1:10 in PBS pH 7.4, and purifiedusing MabSelect Sure Resin (GE Healthcare, Chicago, Ill., USA), followedby size exclusion chromatography. Purified material was quantified usinga Nanodrop™ UV visible spectrophotomer (using a calculated extinctioncoefficient based on the amino acid sequence of the Fc used in theconjugation, and concentrated to approximately 10 mg/mL using acentrifugal concentrator (10,000 MWCO). Purified molecules were analyzedusing 4-12% Bis Tris SDS PAGE gels by loading 1-2 μg of each moleculeinto the gel, and staining using instant Blue staining. Each gelincluded a molecular weight ladder with the indicated molecular weightstandards. Yields are typically 40-60%. MALDI MS analysis showed a rangeof masses (60000-90000) with an average of mass of 63633. AverageDAR=4.5.

Example 33. Efficacy of Conjugate 6 in a Lethal Mouse Influenza Model:Study #3

Conjugate 6 was evaluated against a lethal INFV A H1N1 influenzainfection in female BALB/c mice. The experiment comprised 7 groups of 5mice. At day 0, all mice were challenged with 1×LD90 H1N1 A/Texas/36/91.Groups 1-6 received treatment IV, 28 days before challenge (Table 12).Vehicle (PBS) was included as an additional negative control. Group 7received Oseltamivir phosphate by way of oral delivery, starting 8 hourspost infection twice daily for 5 days. All mice were monitored forsurvival (FIGS. 31A-31F) for 15 days after challenge.

Mice treated with Conjugate 6 showed 100% survival with single doses inall the concentrations down to 2.5 mg/kg, and 80% survival at 1.25mg/kg, compared with 0% survival in the vehicle control group. All micein the Oseltamivir phosphate control group survived. When compared tothe Oseltamivir phosphate group, Conjugate 6 demonstrated similarefficacy to Oseltamivir at a 20× lower cumulative dose (in mg/kg),despite the fact that all Conjugate 6 groups were dosed once, 28 daysprior to infection.

TABLE 12 Study design Group Challenge Dose Treatment n = 5 Day 0Compound (mg/kg) Route/Schedule 1 Influenza A virus, Vehicle (PBS) N/AIV, q.d. 28 days 2 H1N1 strain Conjugate 6 50 pre-challenge 3A/Texas/36/91 by Conjugate 6 10 4 way of IN route. Conjugate 6 5 5Conjugate 6 2.5 6 Conjugate 6 1.25 7 Oseltamivir 20 PO, b.i.d. 8 hours(Tamiflu ™) after challenge for 5 days

Example 34. Efficacy of Conjugate 6 in a Lethal Mouse Influenza Model:Study #4

Conjugate 6 was evaluated against a lethal INFV A H1N1 influenzainfection in female BALB/c mice. The experiment comprised 13 groups of 5mice. At day 0, all mice were challenged with 1×LD90 H1N1 A/Texas/36/91.All Conjugate 6 groups (8-13) received single 10 mg/kg IV doses atdifferent times, pre- and post-infection, as outlined in Table 13.Vehicle (PBS) and Fc only were included as negative controls. Groups 3-7received Oseltamivir phosphate (20 mg/kg) by way of oral delivery,starting at different time points post-infection twice daily for 5 days,as outlined in Table 13. All mice were monitored for survival (FIGS.32A-32F) for 15 days after challenge.

Mice treated with Conjugate 6 showed 100% survival with single 10 mg/kgdoses when treated out to 24 hrs post-infection, and 60 and 80% survivalat 48 and 72 hrs post-infection, respectively. In the Oseltamivirphosphate group, survival dropped sharply to 0% and 20%, respectively,in the groups where treatment was initiated at 48 and 72 hrspost-infection. These results suggest that Conjugate 6 potentiallypossesses a superior treatment window versus Oseltamivir for treatinginfluenza A infections.

TABLE 13 Study design Influenza Dose Group A Dose timing (N = 5) strainTest Article Route, Schedule (mg/kg) (hours)  1 A/Texas/ Vehicle (PBS)IV, single — T − 4  2 36/ Fc only IV, single 10 T − 4  3 91 (H1N1)Oseltamivir PO, bid × 5 days 20 T + 8  4 by way of Oseltamivir PO, bid ×5 days 20 T + 24  5 IN Oseltamivir PO, bid × 5 days 20 T + 48  6Oseltamivir PO, bid × 5 days 20 T + 72  7 Oseltamivir PO, bid × 5 days20 T + 96  8 Conjugate 6 IV, single 10 T − 4  9 Conjugate 6 IV, single10 T + 8 10 Conjugate 6 IV, single 10 T + 24 11 Conjugate 6 IV, single10 T + 48 12 Conjugate 6 IV, single 10 T + 72 13 Conjugate 6 IV, single10 T + 96

Example 35. Conjugate 6 Toxicity Study

The safety of Conjugate 6 was evaluated in a 14 day rat dose-rangefinder toxicity study. Rats were administered either 5 mpk, 20 mpk, or50 mpk of Conjugate 6 by intravenous administration on days 0 and 7 ofthe study. Compared with vehicle controls, no significant effects onbody weight gain (FIG. 33), organ weights, food consumption wereobserved at any dose tested. Plasma exposures (measured by AUC)increased proportionally with dose. These preclinical safety results areconsistent with a high therapeutic index. A summary of observations isprovided in Table 14.

TABLE 14 Summary of 14 day rat dose-range finder toxicity study Findingsat highest dose (50 mpk), Parameter compared to vehicle Clinicalobservations No findings Hematology No change from vehicle ClinicalChemistry No change from vehicle Coagulation No change from vehicleUrinalysis No change from vehicle Histopathology No observations

Example 36. Efficacy of Conjugate 6 Against in a Lethal Mouse InfluenzaModel Dose by Three Different Routes: Study #5

Conjugate 6 was evaluated against a lethal IAV H1N1 influenza infectionin female BALB/c mice (Charles River Laboratories, 6-8 weeks). Thechallenge virus (A/Texas/36/91) is a mouse-adapted isolate capable ofcausing lethal infections in mice. The experiment comprised 15 groups of5 mice. At day 0, all mice were challenged with virus at 1× the LD90 byintranasal inoculation in a volume of 50 μl, after being lightlyanesthetized with isoflurane. Groups 1-14 received a single treatment, 4hours before challenge (Table 15). In order to determine the potency ofConjugate 6 by different dose routes matching concentrations ofConjugate (10, 2, 0.4, and 0.1 mg/kg) were dosed either IV, IM, or SC.In addition to the vehicle (PBS) only group, Human IgG1 (Fc alone) wasincluded as an additional negative control (group 2). Group 15 receivedOseltamivir phosphate by way of oral delivery, starting 8 hours postinfection twice daily for 5 days. All mice were monitored for survival(Table 15) for 14 days.

Mice treated with Conjugate 6 showed 100% survival at day 14 againstchallenge by influenza (H1N1) with single doses of 10, 2, or 0.4 mg/kg,regardless of dose route. Only at the lowest test article concentrationwere single mouse differences seen between IV, IM, and SC dosing (Table16; 60, 80, and 100% survival, respectively). These results stronglysuggest that protective lung concentrations of Conjugate 6 is achievableby multiple dose routes.

TABLE 15 Study design Group Challenge Dose Dose Treatment n = 5 Day 0Compound (mg/kg) route Route/Schedule  1 Influenza A Vehicle (PBS) N/ASingle dose,  2 virus Fc alone 50 IV 4 hours before  3 (H1N1) Conjugate6 10 IV viral challenge  4 A/Texas/ Conjugate 6 2  5 36/91 Conjugate 60.4  6 by way Conjugate 6 0.1  7 of IN Conjugate 6 10 IM  8 Conjugate 62  9 Conjugate 6 0.4 10 Conjugate 6 0.1 11 Conjugate 6 10 SC 12Conjugate 6 2 13 Conjugate 6 0.4 14 Conjugate 6 0.1 15 Oseltamivir 20 POb.i.d. 8 hours (Tamiflu ™) after challenge for 5 days

TABLE 16 Mouse survival % Survival Test article mg/kg Dose route (Day14) Vehicle (PBS) na IV 0 hIgG1 (Fc only) 10 IV 0 Conjugate 6 10 IV 1002 100 0.4 100 0.1 60 Conjugate 6 10 IM 100 2 100 0.4 100 0.1 80Conjugate 6 10 SC 100 2 100 0.4 100 0.1 100 oseltamivir 20 PO 100

Example 37. Efficacy of Conjugate 6 Against an Oseltamivir-ResistantIsolate in a Lethal Mouse Influenza Model: Study #6

Conjugate 6 was evaluated against a lethal IAV H1N1 influenza infectionin female BALB/c mice (Charles River Laboratories, 6-8 weeks). Thechallenge virus (A/Perth/261/2009) is a mouse-adapted isolate thatcarries the H275Y mutation resulting in resistance to the neuraminidaseinhibitor oseltamivir. The experiment comprised 10 groups of 5 mice. Atday 0, all mice were challenged with A/Perth/261/2009 (H1N1) at 1× theLD90 by intranasal inoculation in a volume of 50 μl, to mice lightlyanesthetized with isoflurane. Groups 1-9 received a single treatment byIV, 4 hours before challenge (Table 17). In addition to the vehicle(PBS) only group, Human IgG1 (Fc alone) was included as an additionalnegative control. Group 10 received Oseltamivir phosphate by way of oraldelivery, starting 8 hours post infection twice daily for 5 days. Allmice were monitored for survival (FIGS. 34A-34D, Table 18) and weightloss (FIG. 35, Table 19) for 15 days after challenge.

Mice treated with Conjugate 6 showed 100% survival against challenge byinfluenza (A/Perth/261/2009) with single doses at 50, 10, and 2 mg/kg.Furthermore, despite the small group size (n=5) these results werestatistically significant relative to the vehicle control (Table 18).

At Conjugate 6 concentrations of 0.4, 0.2, or 0.1 mg/kg survival was60%, while no mice survived to the end of the study if dosed withvehicle (PBS) or hIgG1 (Fc only) only. The oseltamivir group only had asingle animal surviving (20% efficacy) despite treatment with a doseshown to be protective against oseltamivir sensitive isolates previously(20 mg/kg, bid×5 days). These results confirm that the challenge virusis resistant to oseltamivir, and sensitive to Conjugate 6.

The potency of Conjugate 6 against mutants containing the H275Y mutationwas further supported by body weight data (FIG. 35, Table 19). Groupsreceiving a single dose of Conjugate 6 at concentrations of 2 mg/kg ormore demonstrated 5%, or less, transient weight loss.

TABLE 17 Study design Group Challenge Dose Treatment n = 5 Day 0Compound (mg/kg) Route/Schedule  1 Influenza A virus Vehicle (PBS) N/AIV, q.d. 4 hours  2 (H1N1) Fc alone 50 pre-challenge  3 A/Perth/261/2009Conjugate 6 50  4 by Conjugate 6 10  5 way of IN route. Conjugate 6 2  6Conjugate 6 0.4  7 Conjugate 6 0.2  8 Conjugate 6 0.1  9 Conjugate 60.05 10 Oseltamivir 20 PO, b.i.d. 8 hours (Tamiflu ™) after challengefor 5 days

TABLE 18 Mouse survival Median Significance Compound Dosage Survival %Survival to vehicle (p) Vehicle (PBS) N/A 7  0 N/A Fc alone   50 mg/kg 6 0 ns Conjugate 6   50 mg/kg Undef 100 0.0027 Conjugate 6   10 mg/kgUndef 100 0.0027 Conjugate 6   2 mg/kg Undef 100 0.0027 Conjugate 6  0.4mg/kg Undef  60 0.1167 Conjugate 6  0.2 mg/kg Undef  60 0.0185 Conjugate6  0.1 mg/kg Undef  60 0.1047 Conjugate 6 0.05 mg/kg 7  0 0.9241Oseltamivir 20 mg/ 7  20 0.3470 phosphate kg B.I.D

TABLE 19 Daily Weight Average Daily average weight (# of mice shown inparenthesis if less than 5) Fc Oseltamivir Days post Vehicle aloneConjugate 6 phosphate infection (PBS) 50 mg/kg 50 mg/kg 10 mg/kg 2 mg/kg0.4 mg/kg 0.2 mg/kg 0.1 mg/kg 0.05 mg/kg 20 mg/kg 0 100 (5) 100 (5) 100100 100 100 100 100 100 100 1 103 (5) 104 (5) 101 101 102 102 103 105102 105 2 102 (5) 105 (5) 103 101 103 100 102 102 101 102 3 93 (5) 97(5) 101 101 98 92 98 95 94 95 4 88 (5) 90 (5) 95 98 99 91 98 92 90 93 585 (5) 85 (5) 94 98 98 91 98 92 89 92 6 83 (4) 88 (3) 100 100 99 88 9585 84 (4) 86 7 79 (3) 90 (2) 103 99 95 90 (3) 94 83 (4) 77 (1) 86 (3) 875 (2) 81 (1) 102 99 97 93 (3) 94 (4) 87 (3) 78 (1) 85 (2) 9 106 104 104101 (3) 101 (4) 95 (3) 86 (2) 10 105 102 103 100 (3) 99 (4) 97 (3) 95(1) 11 106 102 104 102 (3) 104 (4) 102 (3) 97 (1) 12 108 104 105 102 (3)105 (3) 107 (3) 104 (1) 13 107 103 105 102 (3) 105 (3) 107 (3) 104 (1)14 107 103 105 102 (3) 106 (3) 108 (3) 104 (1) 15 100 100 100 100 (3)100 (3) 100 (3) 100 (1)

Example 38. 7-Day Rat PK Study Following IV Administration of TestArticle

Rat pharmacokinetic (PK) studies were performed by Seventh WaveLaboratories (Maryland Heights, Mo.) using male Sprague Dawley Rats46-49 days of age. Rats were injected IV by way of the tail vein with 75mg/kg of test article (5 ml/kg dose volume). Animals were housed understandard IACUC approved housing conditions. At appropriate times animalswere non-terminally bled (retro-orbital, cheek, or by tail vein) withblood collected in K₂EDTA tubes to prevent coagulation. Collected bloodwas centrifuged (2,000×g, for 10 minutes) and plasma withdrawn foranalysis of test article concentrations overtime.

The plasma concentrations for Conjugate 6 or hIgG1 Fc at each time pointwere measured by sandwich ELISA as follows: Conjugate 6 molecules werecaptured either on neuraminidase coated plates or anti-hIgG1 antibodycoated plates and then detected using an HRP-conjugated anti-humanIgG-Fc antibody. hIgG1 was captured using anti-hIgG1 Fc antibody.Protein concentration was calculated in GraphPad Prism using 4PLnon-linear regression of Conjugate 6 (or hIgG1 Fc) standard curves. Amore detailed method description is provided below.

Nunc Maxisorp 96-well plates (Cat No. 12-565-136, ThermoFisher) werecoated with either neuraminidase from A/California/04/2009 (H1N1)(11058-VNAHC, Sino Biological) or anti-IgG1 Fc antibody in 1×KPL coatingbuffer (5150-0041, SeraCare). In the cases where the anti-IgG1 Fcantibody was used to capture test article, capture and detectionanti-IgG1 Fc antibodies were selected that bind different epitopes.Plates were incubated at room temperature for 1 hr on an orbital plateshaker (500 rpm). Serial dilutions of the plasma samples were plated andincubated at room temperature for 2 hrs (sample diluent: 1% BSA in PBS0.05% Tween 20+naïve rat plasma final concentration of 1:900). Conjugate6 or hIgG1 Fc standard curves ranging from 500-0.230 ng/mL, in duplicatewere run on each plate.

Following the 2 hr incubation, plates were washed 5× in 300 uL PBS with0.05% Tween 20. Conjugate 6 bound to neuraminidase on the plates (oranti-hIgG1 Fc antibody) was then probed with an HRP conjugatedanti-human IgG Fc F(ab′)₂ (709-036-098, Jackson) diluted 1:1,000 insample diluent for 1 hr at room temp. Plates were then washed 8× in 300uL PBS with 0.05% Tween 20 and developed with TMB substrate for 7-8minutes. The reaction was stopped with 1N H₂SO₄. Absorbance was read at450 nm. A similar protocol was used for hIgG1 Fc, where only anti-hIgG1Fc antibody was used for capture. The quantities of Conjugate 6 measuredat different time points using either neuraminidase or anti-hIgG1 Fcantibody capture were similar within experimental error, suggesting thatthe intact conjugate is stable in vivo.

Total Conjugate 6 (or hIgG1 Fc) in test samples was interpolated usingin Graphpad Prism Version 6 following nonlinear regression analysis(Sigmoidal, 4PL analysis) of the Conjugate 6 (or hIgG1 Fc) standardcurves. The curves comparing Conjugate 6 and hIgG1 Fc are shown in FIG.36. The quantities of Conjugate 6 measured at different time pointsusing either neuraminidase or anti-hIgG1 Fc antibody capture weresimilar within experimental error, suggesting that the intact conjugateis stable in vivo.

Example 39. 14-Day Rat PK Study Following IV Administration of TestArticle

Rat PK studies were performed by Seventh Wave Laboratories (MarylandHeights, Mo.) using male Sprague Dawley Rats 46-49 days of age. Ratswere injected IV by way of the tail vein with 5, 20, or 50 mg/kg of testarticle (5 ml/kg dose volume) at days 1 and 8. Animals were housed understandard IACUC approved housing conditions. At appropriate times animalswere non-terminally bled (retro-orbital, cheek, or by tail vein) withblood collected in K₂EDTA tubes to prevent coagulation. Collected bloodwas centrifuged (2,000×g, for 10 minutes) and plasma withdrawn foranalysis of test article concentrations overtime.

The plasma concentrations for Conjugate 6 at each time point weremeasured by sandwich ELISA as follows: Conjugate 6 molecules werecaptured either on neuraminidase coated plates and then detected usingan HRP-conjugated anti-human IgG-Fc antibody. hIgG1 was captured usinganti-hIgG1 Fc antibody. Protein concentration was calculated in GraphPadPrism using 4PL non-linear regression of Conjugate 6 standard curves. Amore detailed method description is provided below. Nunc Maxisorp96-well plates (Cat No. 12-565-136, ThermoFisher) were coated witheither neuraminidase from A/California/04/2009 (H1N1) (11058-VNAHC, SinoBiological) in 1X KPL coating buffer (5150-0041, SeraCare). Plates wereincubated at room temperature for 1 hr on an orbital plate shaker (500rpm). Serial dilutions of the plasma samples were plated and incubatedat room temperature for 2 hrs (sample diluent: 1% BSA in PBS 0.05% Tween20+naïve rat plasma final concentration of 1:900). Conjugate 6 standardcurves ranging from 500-0.230 ng/mL, in duplicate were run on eachplate.

Following the 2 hr incubation, plates were washed 5× in 300 uL PBS with0.05% Tween 20. Conjugate 6 bound to neuraminidase on the plates wasthen probed with an HRP conjugated anti-human IgG Fc F(ab′)2(709-036-098, Jackson) diluted 1:1,000 in sample diluent for 1 hr atroom temp. Plates were then washed 8× in 300 uL PBS with 0.05% Tween 20and developed with TMB substrate for 7-8 minutes. The reaction wasstopped with 1N H₂SO₄. Absorbance was read at 450 nm.

Total Conjugate 6 (or hIgG1 Fc) in test samples was interpolated usingin Graphpad Prism Version 6 following nonlinear regression analysis(Sigmoidal, 4PL analysis) of the Conjugate 6 standard curves. The curvescomparing Conjugate 6 shown in FIG. 37 demonstrate linear doseproportionality.

Example 40. 28-Day Rat PK Study Comparing IV and SC Administration ofTest Article

Rat PK studies were performed by Seventh Wave Laboratories (MarylandHeights, Mo.) using male Sprague Dawley Rats 46-49 days of age. Ratswere injected IV or SC with 5 mg/kg of test article (5 ml/kg dosevolume). Animals were housed under standard IACUC approved housingconditions. At appropriate times animals were non-terminally bled(retro-orbital, cheek, or by tail vein) with blood collected in K₂EDTAtubes to prevent coagulation. Collected blood was centrifuged (2,000×g,for 10 minutes) and plasma withdrawn for analysis of test articleconcentrations overtime.

The plasma concentrations for Conjugate 6 or hIgG1 Fc at each time pointwere measured by sandwich ELISA as follows: Conjugate 6 molecules werecaptured on neuraminidase coated plates and then detected using anHRP-conjugated anti-human IgG-Fc antibody. Protein concentration wascalculated in GraphPad Prism using 4PL non-linear regression ofConjugate 6 standard curves. A more detailed method description isprovided below.

Nunc Maxisorp 96-well plates (Cat No. 12-565-136, ThermoFisher) werecoated with either neuraminidase from A/California/04/2009 (H1N1)(11058-VNAHC, Sino Biological) in 1×KPL coating buffer (5150-0041,SeraCare). Plates were incubated at room temperature for 1 hr on anorbital plate shaker (500 rpm). Serial dilutions of the plasma sampleswere plated and incubated at room temperature for 2 hrs (sample diluent:1% BSA in PBS 0.05% Tween 20+naïve rat plasma final concentration of1:900). Conjugate 6 standard curves ranging from 500-0.230 ng/mL, induplicate were run on each plate.

Following the 2 hr incubation, plates were washed 5× in 300 uL PBS with0.05% Tween 20. Conjugate 6 bound to neuraminidase on the plates wasthen probed with an HRP conjugated anti-human IgG Fc F(ab′)2(709-036-098, Jackson) diluted 1:1,000 in sample diluent for 1 hr atroom temp. Plates were then washed 8× in 300 uL PBS with 0.05% Tween 20and developed with TMB substrate for 7-8 minutes. The reaction wasstopped with 1N H₂SO₄. Absorbance was read at 450 nm.

Total Conjugate 6 (or hIgG1 Fc) in test samples was interpolated usingin Graphpad Prism Version 6 following nonlinear regression analysis(Sigmoidal, 4PL analysis) of the Conjugate 6 standard curves. The curvescomparing Conjugate 6 in FIG. 38 show that SC and IV plasma levelsconverged at 24 h and were similar within experimental error out to 336h.

Example 41. 28-Day Non-Human Primate PK Study Following IVAdministration of Test Article

Non-human primate (NHP) PK studies were performed by BTS Research (SanDiego, Calif.) using male and female Cynomolgus monkeys 4.5-8 years oldwith body weights ranging from 2.5-6.5 kg. NHPs were injected IV by wayof the saphenous or cephalic vein with 5 or 20 mg/kg of test article (5ml/kg dose volume). Animals were housed under standard IACUC approvedhousing conditions. At appropriate times animals were non-terminallybled (by way of femoral or cephalic veins) with blood collected inK₂EDTA tubes to prevent coagulation. Collected blood was centrifuged(2,000×g, for 10 minutes) and plasma withdrawn for analysis of testarticle concentrations overtime.

The plasma concentrations for Conjugate 6 at each time point weremeasured by sandwich ELISA as follows: Conjugate 6 molecules werecaptured on neuraminidase coated plates and then detected using anHRP-conjugated anti-human IgG-Fc antibody. Protein concentration wascalculated in GraphPad Prism using 4PL non-linear regression ofConjugate 6 standard curves. A more detailed method description isprovided below.

Nunc Maxisorp 96-well plates (Cat No. 12-565-136, ThermoFisher) werecoated with neuraminidase from A/California/04/2009 (H1N1) (11058-VNAHC,Sino Biological) in 1×KPL coating buffer (5150-0041, SeraCare). Plateswere incubated at room temperature for 1 hr on an orbital plate shaker(500 rpm). Serial dilutions of the plasma samples were plated andincubated at room temperature for 2 hrs (sample diluent: 1% BSA in PBS0.05% Tween 20+naïve cynomolgus monkey plasma final concentration of1:2,500). Conjugate 6 standard curves ranging from 500-0.230 ng/mL, induplicate were run on each plate.

Following the 2 hr incubation, plates were washed 5× in 300 uL PBS with0.05% Tween 20. Conjugate 6 bound to neuraminidase on the plates wasthen probed with an HRP conjugated anti-human IgG Fc F(ab′)2(709-036-098, Jackson) diluted 1:1,000 in sample diluent for 1 hr atroom temp. Plates were then washed 8× in 300 uL PBS with 0.05% Tween 20and developed with TMB substrate for 7-8 minutes. The reaction wasstopped with 1N H₂SO₄. Absorbance was read at 450 nm.

Total Conjugate 6 in test samples was interpolated using in GraphpadPrism Version 6 following nonlinear regression analysis (Sigmoidal, 4PLanalysis) of the Conjugate 6 (or hIgG1 Fc) standard curves. PKparameters were calculated using WinNonlin software. The curvescomparing Conjugate 6 are shown in FIG. 39 and a summary of key PKparameters is provided in Table 20. The dose response is linear between5 and 20 mg/kg IV, resulting in a half-life of approximately 9 daysacross both doses (comparable to mouse/rat).

TABLE 20 Cynomolgus monkey PK of Conjugate 6 Conjugate 6 Half-life (hrs)AUClast (hr*mg/mL)  5 mg/kg IV 230 6240 20 mg/kg IV 197 25400

Example 42. Mouse Lung Distribution PK Study Following IV Administrationof Test Article

Mouse PK studies were performed using male CD-1 mice 6 weeks of age.Mice were injected IV by way of the tail vein with 10 mg/kg of testarticle (5 ml/kg dose volume). Animals were housed under standard IACUCapproved housing conditions. At the indicated time points, the animalswere euthanized to harvest blood (by way of cardiac puncture) in K₂EDTAtubes and lungs. Blood was centrifuged (2,000×g, for 10 minutes) toobtain plasma. The lungs were weighed and homogenized in 1.5 ml tubeswith a sterile disposable pestle (Z359947, Sigma) in 100 uL ofhomogenization buffer comprised of 11.64 mL tissue protein extractionreagent (78510, Thermo Scientific), 0.24 ml of protease inhibitorcocktail (78410, Thermo Scientific), and 0.12 ml of EDTA. After 1-2 minhomogenization, the volume was adjusted to 1 mL with homogenizationbuffer and the samples incubated on ice for 20 min with periodic mixingby gentle vortexing. The homogenates were centrifuged at 8,000×g for 10min and the supernatant retained for analysis of test articleconcentrations overtime.

The plasma and lung concentrations for Conjugate 6 at each time pointwere measured by sandwich ELISA as follows: Conjugate 6 molecules werecaptured on neuraminidase coated plates and detected using anHRP-conjugated anti-human IgG-Fc antibody. Protein concentration wascalculated in GraphPad Prism using 4PL non-linear regression ofConjugate 6 standard curves. A more detailed method description isprovided below.

Nunc Maxisorp 96-well plates (Cat No. 12-565-136, ThermoFisher) werecoated with neuraminidase from A/California/04/2009 (H1N1) (11058-VNAHC,Sino Biological) in 1×KPL coating buffer (5150-0041, SeraCare). Plateswere incubated at room temperature for 1 hr on an orbital plate shaker(500 rpm). Serial dilutions of the plasma samples were plated andincubated at room temperature for 2 hrs (sample diluent: 1% BSA in PBS0.05% Tween 20+naïve mouse plasma final concentration of 1:100).Conjugate 6 standard curves ranging from 500-0.230 ng/mL, in duplicatewere run on each plate.

Following the 2 hr incubation, plates were washed 5× in 300 uL PBS with0.05% Tween 20. Conjugate 6 bound to neuraminidase on the plates wasthen probed with an HRP conjugated anti-human IgG Fc F(ab′)2(709-036-098, Jackson) diluted 1:1,000 in sample diluent for 1 hr atroom temp. Plates were then washed 8× in 300 uL PBS with 0.05% Tween 20and developed with TMB substrate for 7-8 minutes. The reaction wasstopped with 1N H₂SO₄. Absorbance was read at 450 nm.

Total Conjugate 6 in test samples was interpolated using in GraphpadPrism Version 6 following nonlinear regression analysis (Sigmoidal, 4PLanalysis) of the Conjugate 6 standard curves. The curves comparingConjugate 6 plasma and lung concentrations are shown in FIG. 40.Unexpectedly, lung C_(max) was reached a tt=1 h (19.5 μg/g lung tissue,˜310 nM). There was approximately 10% lung exposure of Conjugate 6,relative to plasma

Example 43. 5-Day Mouse PK Study Comparing IV, SC and IM Administrationof Test Article

Mouse PK studies were performed using male CD-1 mice 6 weeks of age.Mice were injected IV byway of the tail vein with 5 mg/kg of testarticle (5 ml/kg dose volume). Animals were housed under standard IACUCapproved housing conditions. At appropriate times animals werenon-terminally bled (retro-orbital, cheek, or by tail vein) with bloodcollected in K₂EDTA tubes to prevent coagulation. Collected blood wascentrifuged (2,000×g, for 10 minutes) and plasma withdrawn for analysisof test article concentrations overtime.

The plasma concentrations for Conjugate 6 at each time point weremeasured by sandwich ELISA as follows: Conjugate 6 molecules werecaptured on neuraminidase coated plates and then detected using anHRP-conjugated anti-human IgG-Fc antibody. Protein concentration wascalculated in GraphPad Prism using 4PL non-linear regression ofConjugate 6 (or hIgG1 Fc) standard curves. A more detailed methoddescription is provided below.

Nunc Maxisorp 96-well plates (Cat No. 12-565-136, ThermoFisher) werecoated with neuraminidase from A/California/04/2009 (H1N1) (11058-VNAHC,Sino Biological) in 1×KPL coating buffer (5150-0041, SeraCare). Plateswere incubated at room temperature for 1 hr on an orbital plate shaker(500 rpm). Serial dilutions of the plasma samples were plated andincubated at room temperature for 2 hrs (sample diluent: 1% BSA in PBS0.05% Tween 20+naïve mouse plasma final concentration of 1:100).Conjugate 6 standard curves ranging from 500-0.230 ng/mL, in duplicatewere run on each plate.

Following the 2 hr incubation, plates were washed 5× in 300 uL PBS with0.05% Tween 20. Conjugate 6 bound to neuraminidase on the plates wasthen probed with an HRP conjugated anti-human IgG Fc F(ab′)2(709-036-098, Jackson) diluted 1:1,000 in sample diluent for 1 hr atroom temp. Plates were then washed 8× in 300 uL PBS with 0.05% Tween 20and developed with TMB substrate for 7-8 minutes. The reaction wasstopped with 1N H₂SO₄. Absorbance was read at 450 nm.

Total Conjugate 6 in test samples was interpolated using in GraphpadPrism Version 6 following nonlinear regression analysis (Sigmoidal, 4PLanalysis) of the Conjugate 6 standard curves. The curves comparingConjugate 6 are shown in FIG. 41. The exposure levels for IV, IM and SCwere similar with AUCs of 2082, 1944 and 2500, respectively.

Example 44. Mouse Efficacy and Multi-Species PK Supports InfrequentProphylactic Dosing in Human Subjects

Allometric scaling of mouse efficacy dosing based on mouse, rat, andprimate PK studies was used to predict dosing and PK parameters in humansubjects. Allometric scaling was based on the Area Under the Curve (AUC)at a 2.5 mg/kg efficacious dose in a 28-day mouse prevention study (seeExample 33).

For cynomolgus monkey-only allometric scaling, human clearance (CL) wascalculated based on cynomolgus monkey PK data (Example 41) only using asimple allometric equation:CL(human)=CL(monkey)·[BW(human)/BW(monkey)]^(w), where BW=bodyweight andw is the scaling exponent fixed at 0.85. The results of cynomolgusmonkey-only scaling are provided in Table 21.

For Mouse-rat-cynomolgus allometric scaling, human clearance (CL) fromanimal species was plotted against the animal body weight (BW) on alog-log scale according to the following allometric equation:CL=a*BW^(x), where a is the coefficient and x is the exponent of theallometric equation. The coefficient a and exponent x were calculatedfrom the intercept and slope of the linear regression line,respectively. The results of mouse-rat-cyno scaling are provided inTable 22.

Human clearance values calculated by respective algorithms above werethen used to calculate the corresponding human dose needed to achievethe Efficacy AUC target of 3700 ug-hr/mL (from mouse 2.5 mg/kg dose,Example 33) using the following equation: Dose=CL*AUC.

TABLE 21 Cynomolgus monkey-only allometric scaling (β = 0.85) EfficacyArea Under the Curve (AUC) 3700 μg-hr/mL Human Clearance (CL) 0.43mL/min Human Dose 95.46 mg, 1.4 mg/kg

TABLE 22 Mouse-rat-cyno allometric scaling (β = 0.97) Efficacy AreaUnder the Curve (AUC) 3700 μg-hr/mL Human Clearance (CL) 0.59 mL/minHuman Dose 130.98 mg, 1.9 mg/kg

Example 45. Synthesis of Int-11

Step a

A solution of the tert-butyl (4-bromobutyl)carbamate (11.2 g, 60 mmol)and tert-butyl (4-aminobutyl)carbamate (10 g, 40 mmol) dissolved in DMF(150 mL) was treated with potassium carbonate (16.4 g, 120 mmol), thenstirred at 80° C. for 6 hrs. The reaction mixture was partitionedbetween DCM (500 ml) and brine (100 ml). The organic layer was separatedand washed with brine then dried with sodium sulfate. The solution wasfiltered, concentrated, and purified by reverse phase liquidchromatography (RPLC) using an Isco COMBIFLASH® liquid chromatographeluted with 10% to 100% acetonitrile and water with 0.1% TFA asmodifier. Yield of the product 11.0 g, 77%. Ion(s) found by LCMS:M+H=360.4

Step b

Product from the previous step (0.4 g, 1.11 mmol) and Fmoc-OSu (0.45 mg,1.3 mmol) were dissolved in DCM (10 mL), then treated withN-methylmorpholine (0.22 ml, 2 mmol), and stirred for 1 hr at roomtemperature. The reaction was concentrated and purified by and purifiedby reverse phase liquid chromatography (RPLC) using an Isco COMBIFLASH®liquid chromatograph eluted with 10% to 100% acetonitrile and water withno 0.1% TFA as modifier. Yield of the products 450 mg, 69%. Ion(s) foundby LCMS: M/2+H=582.4.

Step c

Product from the previous step (0.4 g, 0.7 mmol) was treated with TFA (5mL) at room temperature for 0.5 hour, then concentrated to dryness andused for next step without further purification. Yield was quantitativefor this step, Ion(s) found by LCMS: M/2+H=382.3.

Step d

Fmoc diamine (24 mg, 0.063 mmol) from the previous step was added to asolution of carboxylic acid (80 mg, 0.126 mmol, described in thesynthesis Int-10, step f) in DMF (5 mL), then treated with HATU (50 mg,0.13 mmol), followed N-methylmorpholine (0.06 ml, 0.50 mmol). Afterstirring for 1 h the resulting solution was concentrated and purified byreverse phase liquid chromatography (RPLC) using an Isco COMBIFLASH®liquid chromatograph eluted with 10% to 100% acetonitrile and waterwithout TFA as a modifier. Yield of the products 80 mg, 79%. Ion(s)found by LCMS: M/2+H=803.9.

Step e

Product from the previous step (80 mg, 0.050 mmol) was treated with 1%DBU (1,8-diazabicyclo[5.4.0]undec-7-ene) in DMF (2 mL) and stirred atroom temperature. When the reaction was complete by LCMS (15 min), itwas concentrated, then treated with TFA (2 mL) and stirred for 30 min atroom temperature. The reaction solution was concentrated and purified byreverse phase liquid chromatography (RPLC) using an Isco COMBIFLASH®liquid chromatograph eluted with 10% to 100% acetonitrile and water,using 0.1% TFA as the modifier. Yield of product was 52 mg as TFA salt,Ion(s) found by LCMS: M/2+H=492.7.

Step f

Product from the previous step was dissolved into water (2 mL), thentreated with a lithium hydroxide (12 mg, 0.50 mmol, in 1 mL water)solution. The resulting reaction was monitored by LCMS. After stirringfor 10 min the reaction was quenched with 0.1 ml acetic acid. Theresulting solution was concentrated and purified by reverse phase liquidchromatography (RPLC) using an Isco COMBIFLASH® liquid chromatographeluted with 0% to 30% acetonitrile and water, using 0.1% TFA as themodifier. Yield of the product: 30 mg as TFA salt, 66%. Ion(s) found byLCMS: M/2+H=452.7. M/3+H=302.1.

Example 46. Synthesis of Int-12

Step a

To a solution of tert-butyl (4-bromobutyl)carbamate (4.8 g, 19 mmol) andpropargyl-PEG₄ amine (2 g, 8.6 mmol) in DMF (50 mL) was added potassiumcarbonate (3.6 g, 26 mmol). The solution was stirred at 80° C. for 6hrs, then partitioned between DCM (200 ml) and brine (50 ml). Theorganic layer was separated, washed with brine, dried with sodiumsulfate, filtered, and concentrated, then purified by reverse phaseliquid chromatography (RPLC) using an Isco COMBIFLASH® liquidchromatograph eluted with 10% to 100% acetonitrile and water with 0.1%TFA as modifier. Yield of product 3.5 g, 70%. Ion(s) found by LCMS:M/2+H=574.4.

Step b

Product from the previous step (3.5 g, 8.6 mmol) was treated with TFA(20 ml) at room temperature for 0.5 hour, then concentrated to dryness,dissolved in water, frozen, lyophilized, and used in the next stepwithout further purification. The yield was quantitative for this step,Ion(s) found by LCMS: M/2+H=374.3.

Step c

Diamine TFA salt from the previous step (37 mg, 0.1 mmol) was added to asolution of the carboxylic acid (130 mg, 0.2 mmol, described in thesynthesis Int-10, step f) in 10 ml DMF, then treated with HATU (80 mg,0.2 mmol), and N-methylmorpholine (0.25 ml, 2 mmol). The resultingsolution was stirred for 1 hr, then concentrated and purified by reversephase liquid chromatography (RPLC) using an Isco COMBIFLASH® liquidchromatograph eluted with 10% to 100% acetonitrile and water with no0.1% TFA as modifier. Yield of the product 120 mg, 75%. Ion(s) found byLCMS: M/2+H=799.9.

Step d

Product from the previous step (120 mg, 0.075 mmol) was treated with 2ml trifluoroacetic acid and stirred for 30 min at room temperature. Theresulting solution was concentrated, dissolved into water (2 mL), thentreated with a solution of lithium hydroxide (12 mg, 0.5 mmol) dissolvedin water (1 mL). The resulting solution was stirred 10 min, then madeslightly acidic with 0.1 ml acetic acid, concentrated and purified byreverse phase liquid chromatography (RPLC) using an Isco COMBIFLASH®liquid chromatograph eluted with 0% to 30% acetonitrile and water, using0.1% TFA as the modifier. Yield of the products 48 mg as TFA salt, 57%.Ion(s) found by LCMS: M/2+H=559.757. M/3+H=373.5.

Example 47. Synthesis of Conjugate 8

A solution of h-IgG1 Fc-PEG4-azide in PBS×1 buffer solution (100 mg,1.71 μmol, 7.011 mL, MW=58200 Da, DAR=3.7) were added alkyne derivatizedsmall molecule (Int-12 TFA salt, 45 mg, 0.031 mmol) and freshly preparedpH 7.4 PBS solutions of CuSO4 (0.7 mL of 50.0 mM, 20 eq),tris(3-hydroxypropyltriazolylmethyl)-amine (THPTA, 0.7 mL of 50.0 mM, 20eq), and sodium ascorbate (1.05 mL of 50.0 mM, 30 eq). The resultinghomogeneous solution was agitated by rocker table for 12 h. The crudesolutions were diluted with pH 7.4 PBS to a final concentration of 1mg/mL, and ultrafiltered (10,000 MWCO) to a volume of 1 mL, two times.The crude mixtures were then diluted 1:10 in PBS pH 7.4, and purifiedusing MabSelect Sure Resin (GE Healthcare, Chicago, Ill., USA), followedby size exclusion chromatography. Purified material was quantified usinga Nanodrop™ UV visible spectrophotomer (using a calculated extinctioncoefficient based on the amino acid sequence of the Fc used in theconjugation, and concentrated to approximately 10 mg/mL using acentrifugal concentrator (10,000 MWCO). Purified molecules were analyzedusing 4-12% Bis Tris SDS PAGE gels by loading 1-2 μg of each moleculeinto the gel, and staining using instant Blue staining. Each gelincluded a molecular weight ladder with the indicated molecular weightstandards (FIG. 42). Yields are typically 40-60%. MALDI MS analysisshowed a range of masses (60000-90000) with an average of mass of 62358.Average DAR=3.

Example 48. Comparison of In Vitro and In Vivo Potency of SelectedInhibitors with their Fc-Conjugates in CPE Assays and in a Lethal MouseInfluenza Model

To demonstrate that conjugation of neuraminidase inhibitors describedherein to Fc enhances their activities in viral replication assays andin in vivo efficacy models, we compared the activities of selectedunconjugated inhibitors to their Fc conjugates in Cytopathic Effect(CPE) assays, and in a lethal mouse influenza infection model. For theCPE microneutralization assay, ten two-fold serial dilutions of eachtest article (TA), starting at 160 nM or 400 nM (9600 nM for zanamivirand oseltamivir controls), were prepared in duplicate for one-hourinoculation with INFV A (A/CA/09 H1N1) at a multiplicity of infection(MOI) 0.001 and INFV B at a MOI of 0.01 for one-hour incubation. TheTA-virus mix was then added to MDCK cells seeded in 96-well plates, andincubated for one hour. On day 3 post infection for INFV A and 5 forINFV B (B/Brisbane), Cells were fixed and stained with crystal violetand optical density was read for calculation of 50 percent effectiveconcentration (EC₅₀) of each TA using XLfit dose response model. Theintrinsic cytotoxicity of each TA was also evaluated using ten two-foldserial dilutions of each TA starting at 160 nM or 400 nM were preparedin duplicate for inoculation with MDCK cells in 96-well culture plates.The cell by way ofbility was determined 3 and 5 days post treatmentusing CellTiter-Glo kit. 50% of cytotoxicity concentration (CC₅₀) wascalculated using XLfit dose response model. No cytotoxicity was observedfor any of the TAs to the highest concentrations tested. A summary ofthe CPE-based microneutralization assay is shown in Table 23.

TABLE 23 CPE-based microneutralization assay EC₅₀ vs EC₅₀ vs INFV A (nM)INFV B (nM) Oseltamivir 390.0 1065.0 Zanamivir 264.8 382.6 Int -7(Targeting Moiety 665.7 106.6 corresponding to Conugate 6) Int-12(Targeting Moiety 15.4 0.6 corresponding to Conjugate 8) Conjugate 6 4.052.1 Conjugate 8 0.6 0.3

The data summarized in Table 23 demonstrate that the Fc-conjugated formsof the neuraminidase dimers possess superior activity in CPEmicroneutralization assay to their unconjugated counterpart.Enhancements of 166-fold and 2-fold, versus INFV A and INFV B,respectively, were observed when comparing Conjugate 6 to Int-7.Enhancements of 26-fold and 2-fold, versus INFV A and INFV B,respectively, were observed when comparing Conjugate 8 to Int-12.

Conjugate 6 was compared to a the most potent neuraminidase inhibitordimer from the study summarized in Table 23 (Int-11, dimer-onlycorresponding to Int-12 without the trimeric linker allowing forconjugation to an Fc) in a lethal IAV H1N1 influenza infection modelusing female BALB/c mice (Charles River Laboratories, 6-8 weeks). Thechallenge virus (A/Puerto Rico/08/1934, aka PR8) is a mouse-adaptedisolate with a LD₉₀ of approximately 30 plaque-forming units (pfu) permouse.

The experiment comprised 8 groups of 5 mice. At day 0, all mice werechallenged with PR8 at 10× the LD₉₀ by intranasal inoculation in avolume of 50 μl, to mice anesthetized with a mixture ofketamine/xylazine (150 and 10 mg/kg respectively). Groups received asingle treatment of vehicle, hIgG1 Fc only, conjugate 6, or Int-11 byIV, 2 hours after challenge (Table 24; groups 1-6). Group 7 receivedInt-11 (15 mg/kg) twice daily (bid) for 5 days, also by IV. Group 8received oseltamivir (15 mg/kg) orally (PO), bid, for 5 days. All micewere monitored for survival (Table 25) and weight loss (data not shown)for 10 days after viral challenge.

As expected, mice treated with vehicle or hIgG1 Fc only succumbed to theinfection by day 7 (Table 25). Mice treated with 10 doses (150 mg/mousein total) of oseltamivir demonstrated a statistically significant delayin death but only a single animal survived until day 10. All micereceiving a single dose of conjugate 6 at 0.3 or 3 mg/kg survived to theend of the study and showed a net weight gain over the course of theexperiment. Importantly, all mice receiving the Int-11 at 0.3 or 3 mg/kgdied over the course of the study, with only a minimal (2 days or less)delay in death relative to the vehicle and hIgG1 Fc only controls. Asthe hIgG1 Fc has no inherent antiviral activity (group 2) this suggeststhe greatly improved activity of conjugate 6 is the result of theimproved avidity resulting from the multivalent display on an Fc, assuggested by the results summarized in Table 23, as well as improvedpharmacokinetics and contribution from Fc mediated immune engagement.The difference in activity between the inhibitor dimer alone andconjugate 6 was statistically significant at both dose concentrations(compare groups 3 and 6, and groups 4 and 5; Table 25). On a mass basis,a 500× greater cumulative dose of Int-11 was required to observeequivalent efficacy to conjugate 6.

TABLE 24 Study design Group Challenge Dose Treatment n = 5 Day 0Compound (mg/kg) Route/Schedule 1 Influenza A Vehicle (PBS) N/A IV,Singe dose 2 virus (H1N1) hlgG1 Fc only 3 starting 2 hours A/Puerto postinfection 3 Rico/08/1934 Conjugate 6 3 4 by way Conjugate 6 0.3 5 of INroute. Int-11 0.3 6 Int-11 3 7 Int-11 15 IV, bid for 5 days starting 2hours post infection 8 Oseltamivir 20 PO, bid for 5 days (Tamiflu ™)starting 2 hours post infection

TABLE 25 Mouse survival Significance to Int-11 Dose Dosing % Signficance0.3 mg/kg* or 3 Group Test article (mg/kg) schedule Survival to vehiclemg/kg{circumflex over ( )} 1 Vehicle (PBS) IV, Single 0 2 hlgG1 Fc only3 IV, Single 0 NS 3 Conjugate 6 3 IV, Single 100 0.00270.0035{circumflex over ( )} 4 Conjugate 6 0.3 IV, Single 100 0.00270.0027* 5 Int-11 0.3 IV, Single 0 0.0027 6 Int-11 3 IV, Single 0 0.00277 Int-11 15 IV, bid × 5 days 100 0.0027 8 Oseltamivir 15 PO, bid × 5days 20 0.0027

Example 49. Synthesis of Int-13((5R)-((1R)-Acetylamino-(2S)-methoxy-(2S)-methylpentyl)-(4S)-E/Z-[3-(propargyl-PEG4)-propenyl]-pyrrolidine-(2R)-carboxylicacid)

Step a.(2R)-((1R)-Acetylamino-(2S)-methoxy-(2S)-methylpentyl)-(5R)-carboxy-(3S)-Z-propenylpyrrolidine-1-carboxylicacid tert-butyl ester

To a stirring mixture of(5R)-((1R)-acetylamino-(2S)-methoxy-(2S)-methylpentyl)-(4S)-Z-propenylpyrrolidine-(2R)-carboxylicacid, HCl salt (prepared accordingly to reference JACS, 2002, 124,4716-4721; 1.0 mmol) in acetonitrile (10 mL) it is addedtrimethylammonium hydroxide (1.5 mmol). After stirring for 3 h at roomtemperature the di-tert-butyldicarbonate (4 eqmol) is added. Uponreaction completion, all the volatiles are evaporated per vacuumtechniques. The residue is diluted with water (10 ml). Ethyl acetate (10ml) is added, and 1 M sulfuric acid aqueous solution is added until thewater layer reaches pH˜3. The water layer is washed with two additionalaliquots of ethyl acetate (10 mL). The combined organics are dried oversodium sulfate, filtered and concentrated. The residue is purified bychromatographic techniques to afford the desired product.

Step b.(2R)-((1R)-Acetylamino-(2S)-methoxy-(2S)-methylpentyl)-(5R)-carboxymethyl-(3S)-Z-propenylpyrrolidine-1-carboxylicacid tert-butyl ester

To a 0° C. stirring mixture of(2R)-((1R)-acetylamino-(2S)-methoxy-(2S)-methylpentyl)-(5R)-carboxy-(3S)-Z-propenylpyrrolidine-1-carboxylicacid tert-butyl ester (1.0 mmol) in dichloromethane (5.0 mL) andmethanol (1.0 mL) it is slowly added (trimethylsilyl)diazomethane (1.1mmol). The mixture is stirred until completion, while temperature isgently allowed to reach ambient. All the volatiles are evaporated pervacuum techniques. If necessary, the residue is purified bychromatographic techniques to afford the desired product.

Step c.(2R)-((1R)-Acetylamino-(2S)-methoxy-(2S)-methylpentyl)-(5R)-carboxymethyl-(3S)-formylpyrrolidine-1-carboxylicacid tert-butyl ester

A room temperature mixture of(2R)-((1R)-acetylamino-(2S)-methoxy-(2S)-methylpentyl)-(5R)-carboxymethyl-(3S)-Z-propenylpyrrolidine-1-carboxylicacid tert-butyl ester (1 mmol) in dichloromethane (15 mL) is cooled to−78° C. Ozone is bubbled through the solution until a faint blue colorof dissolved ozone persists. Nitrogen is bubbled through the solutionuntil blue color disappears, then dimethyl sulfide (4.0 mmol) is added,the flask transferred into a freezer (−20° C.) and let sit for 1 hour.The solution is concentrated and the residue is purified bychromatographic techniques to afford the desired product.

Step d.(2R)-((1R)-Acetylamino-(2S)-methoxy-(2S)-methylpentyl)-(5R)-carboxymethyl-(3S)-E/Z-[3-(propargyl-PEG4)-propenyl]pyrrolidine-1-carboxylicacid tert-butyl ester

To a 0° C. stirring mixture of propargyl-PEG4-phosphonium bromide (1.0mmol) in DMF (5.0 mL) it is added sodium hydride (1.1 mmol), and after10 minutes temperature is raised to ambient. Stirring is continued for 1h, then(2R)-((1R)-Acetylamino-(2S)-methoxy-(2S)-methylpentyl)-(5R)-carboxymethyl-(3S)-formylpyrrolidine-1-carboxylicacid tert-butyl ester (1.0 mmol) is added in DMF (1.0 mL). Uponcompletion, the reaction is quenched by saturated ammonium chloridesolution. The aqueous solution is extracted several times with ethylacetate, and the combined organic phases are washed with brine, dried,and evaporated. The residue is purified by chromatographic techniques toafford the desired product.

Step e.(2R)-((1R)-Acetylamino-(2S)-methoxy-(2S)-methylpentyl)-(5R)-carboxy-(3S)E/Z-[3-(propargyl-PEG4)-propenyl]pyrrolidine-1-carboxylic acidtert-butyl ester

To a 0° C. stirring mixture of(2R)-((1R)-Acetylamino-(2S)-methoxy-(2S)-methylpentyl)-(5R)-carboxymethyl-(3S)-E/Z-[3-(propargyl-PEG4)-propenyl]pyrrolidine-1-carboxylicacid tert-butyl ester (1.0 mmol) in tetrahydrofuran (12.0 mL) and water(3.0 mL) it is added lithium hydroxide (1.1 mmol). Stirring is continuedand the temperature is raised to ambient after 15 minutes. Uponcompletion, the solution is brought to acidic pH by the means of theexcess addition of AMBERLITE® IRN-77 resin. The mixture is filtered andthe filtrate is concentrated per vacuum techniques, yielding to thetitle compound. If necessary, the residue is purified by chromatographictechniques to afford the desired product.

Step f.(5R)-((1R)-Acetylamino-(2S)-methoxy-(2S)-methylpentyl)-(4S)-E/Z-[3-(propargyl-PEG4)-propenyl]-pyrrolidine-(2R)-carboxylicacid

To a 0° C. stirring mixture of(2R)-((1R)-Acetylamino-(2S)-methoxy-(2S)-methylpentyl)-(5R)-carboxy-(3S)E/Z-[3-(propargyl-PEG4)-propenyl]pyrrolidine-1-carboxylic acidtert-butyl ester (1.0 mmol) and 2-methyl-2-butene (0.5 mL) indichloromethane (8.0 mL), it is added trifluoroacetic acid (4.0 mL).After 10 minutes, the temperature is raised to ambient. Upon completion,all the volatiles are evaporated per vacuum techniques. The residue ispurified by chromatographic techniques to afford the desired product.

Example 50. Synthesis of Conjugate 9

A solution of hIgG1 Fc-PEG4-azide in pH 7.4 PBS×1 buffer solution (100mg, 1.71 umol, 7.011 mL, MW=58,200 Da, DAR=3.7) is added to a pH 7.4PBS×1 buffer solution (2.45 mL) of Int-13((5R)-((1R)-Acetylamino-(2S)-methoxy-(2S)-methylpentyl)-(4S)-E/Z-[3-(propargyl-PEG4)-propenyl]-pyrrolidine-(2R)-carboxylicacid) (0.031 mmol), cupric sulfate (0.62 mmol),tris(3-hydroxypropyltriazolylmethyl)-amine (0.62 mmol), and sodiumascorbate (0.93 mmol). The resulting homogeneous solution is gentlyshaken with a rocker table for 12 h. The crude solution is diluted withpH 7.4 PBS to a final concentration of 1 mg/mL, and ultrafiltered(10,000 MWCO) to a volume of 1 mL, for two times. The crude mixture isthen diluted 1:10 in PBS pH 7.4, and purified using MabSelect Sure Resin(GE Healthcare, Chicago, Ill., USA), followed by size exclusionchromatography. Purified material is quantified using a Nanodrop™ UVvisible spectrophotomer (using a calculated extinction coefficient basedon the amino acid sequence of the Fc used in the conjugation, andconcentrated to approximately 10 mg/mL using a centrifugal concentrator(10,000 MWCO). Purified molecules are analyzed using 4-12% Bis Tris SDSPAGE gels by loading 1-2 ug of each molecule into the gel, and stainingusing instant Blue staining. Each gel includes a molecular weight ladderwith the indicated molecular weight standards. MALDI MS analysis is usedto determine the average DAR.

Example 51. Synthesis of Propargyl-PEG4-Phosphonium Bromide

A mixture of propargyl-PEG4-bromide (1.0 mmol) and triphenylphosphine(1.2 mmol) in toluene (10 mL) are refluxed. Upon completion, the mixtureis cooled to ambient. The solids are filtered and used in the next stepwithout any additional purification.

Example 52. Synthesis of Int-14((5R)-[(1R)-(propargyl-PEG4-carboxyamide)-(2S)-methoxy-(2S)-methylpentyl]-(4S)-Z-propenylpyrrolidine-(2R)-carboxylicacid, HCl Salt)

Step a.N-{(2.S)-methoxy-((1R)-[1-(4-methoxybenzyl)-5-oxo-(3S)-Z-propenylpyrrolidin-(2R)-yl]-(2S)-methylpentyl}-(propargyl-PEG4)-carboxyamide

To 0° C. a stirring mixture of{(2S)-Methoxy-(1R)-[1-(4-methoxybenzyl)-5-oxo-(3S)-Z-propenylpyrrolidin-(2R)-yl]-(2S)-methylpentyl}-carbamicacid tert-butyl ester (it is prepared accordingly to reference JACS,2002, 124, 4716-4721; 1.0 mmol) in dry dichloromethane (5 mL) is addedtrifluoroacetic acid (10 mmol), and the temperature is raised toambient. Upon completion, the solvent is removed under reduced pressure.The resulting residue is dissolved in dichloromethane (20 mL) andextracted with a saturated aqueous solution of sodium bicarbonate. Theorganic layer is separated, dried (sodium sulfate), filtered andevaporated. The crude amine is dissolved in DMF (5 mL) and treated at 0°C. under stirring with propargyl-PEG4-acid (1.1 mmol),diisopropylethylamine (3.0 mmol) and HATU (1.1 mmol). Upon reactioncompletion, all the volatiles are evaporated per vacuum techniques. Theresidue is taken up in ethyl acetate (15 ml), and washed with saturatedaqueous solution of sodium bicarbonate (10 mL), then 1 M sulfuric acidaqueous solution (10 mL). The combined organics are dried over sodiumsulfate, filtered and concentrated. The residue is purified bychromatographic techniques to afford the desired product.

Step b.(2R)-((1R)-(propargyl-PEG4-carboxyamide)-(2S)-methoxy-(2S)-methylpentyl)-(5R)-oxo-(3S)-Z-propenylpyrrolidine-1-carboxylicacid tert-butyl ester

To a stirring solution ofN-{(2.S)-methoxy-(1R)-[1-(4-methoxybenzyl)-5-oxo-(3S)-Z-propenylpyrrolidin-(2R)-yl]-(2S)-methylpentyl}-(propargyl-PEG4)-carboxyamide(1.0 mmol) in a mixture of acetonitrile and water (10:1.5 mL) is addedceric ammonium nitrate (2.0 mmol) in small portions at 45° C. during 1h, and stirring is continued until completion. The reaction is quenchedwith a saturated aqueous solution of sodium bicarbonate (5 mL). Theaqueous layer is extracted with EtOAc (3×10 mL), the combined organiclayers are dried (sodium sulfate) and evaporated to give a crude whichis used for the next step without further purification. The material isdissolved in acetonitrile (5 mL), di-tert-butylcarbonate is added (1.5mmol) followed by triethylamine (2.0 mmol) and DMAP (catalytic). Uponcompletion, the reaction is quenched with a saturated solution ofammonium chloride (5 mL). The aqueous layer is extracted with EtOAc(3×10 mL), and the combined organic layers are dried (sodium sulfate).All the volatiles are removed per vacuum techniques. If necessary, theresidue is purified by chromatographic techniques to afford the desiredproduct.

Step c.(2R)-((1R)-(propargyl-PEG4-carboxyamide)-(2S)-methoxy-(2S)-methylpentyl)-(5R/S)-methoxy-(3S)-Z-propenylpyrrolidine-1-carboxylicacid tert-butyl ester

To a −78° C. stirring solution of(2R)-((1R)-(propargyl-PEG4-carboxyamide)-(2S)-methoxy-(2S)-methylpentyl)-(5R)-oxo-(3S)-Z-propenylpyrrolidine-1-carboxylicacid tert-butyl ester (1.0 mmol) in THF (8 mL) is added SUPER-HYDRIDE®(1 M in THF, 2.2 mmol). After 30 min the reaction mixture is quenchedwith a saturated aqueous solution of sodium bicarbonate (4 mL) and 30%hydrogen peroxyde (5 drops). The mixture is warmed up to rt and stirredfor another 30 min and the aqueous layer is extracted with EtOAc (3×10mL). The combined organic layers are dried (sodium sulfate) and thesolvent is evaporated to give the hemiaminal, which is used withoutfurther purification. To a solution of the above product in methanol (16mL) is added p-toluenesulfonic acid hydrate (0.1 mmol) at rt. Thereaction mixture is stirred overnight and is quenched with a saturatedaqueous solution of sodium bicarbonate (10 mL). Methanol is removedunder reduced pressure, water (10 mL) is added to the resulting residueand extracted with EtOAc (3×10 mL). The organics are separated and driedwith brine and sodium sulfate, filtered and concentrated. If necessary,the residue is purified by chromatographic techniques to afford thedesired product.

Step d.(2R)-((1R)-(propargyl-PEG4-carboxyamide)-(2S)-methoxy-(2S)-methylpentyl)-(5R)-cyano-(3S)-Z-propenylpyrrolidine-1-carboxylicacid tert-butyl ester

To a −78° C. stirring solution of(2R)-((1R)-(propargyl-PEG4-carboxyamide)-(2S)-methoxy-(2S)-methylpentyl)-(5R/S)-methoxy-(3S)-Z-propenylpyrrolidine-1-carboxylicacid tert-butyl ester (1.0 mmol) in dichloromethane (20 mL) it is addedtrimethylsilyl cyanide (2.0 mmol) followed by boron trifluoride diethyletherate (1.2 mmol). The reaction mixture is stirred from −78° C. to−50° C. over a period of 3 h. A saturated aqueous solution of sodiumbicarbonate (40 mL) is added and the aqueous layer is extracted withEtOAc (3×15 mL). The combined organic layers are dried (sodium sulfate),filtered and concentrated under reduced pressure. The resulting residueconsisting of a mixture of epimeric cyano derivatives is purified bychromatographic techniques to afford the desired product.

Step e.(5R)-[(1R)-(propargyl-PEG4-carboxyamide)-(2S)-methoxy-(2S)-methylpentyl]-(4S)-Z-propenylpyrrolidine-(2R)-carboxylicacid, HCl Salt

To a solution of(2R)-((1R)-(propargyl-PEG4-carboxyamide)-(2S)-methoxy-(2S)-methylpentyl)-(5R)-cyano-(3S)-Z-propenylpyrrolidine-1-carboxylicacid tert-butyl ester (1.0 mmol) in AcOH (10 mL), 12N HCl (10 mL) isadded at rt. The solution is stirred at rt until completion, the solventis evaporated under reduced pressure. If necessary, the residue ispurified by chromatographic techniques to afford the desired product.

Example 53. Synthesis of Conjugate 10

A solution of hIgG1 Fc-PEG4-azide in pH 7.4 PBS×1 buffer solution (100mg, 1.71 umol, 7.011 mL, MW=58,200 Da, DAR=3.7) is added to a pH 7.4PBS×1 buffer solution (2.45 mL) of Int-14((5R)-[(1R)-(propargyl-PEG4-carboxyamide)-(2S)-methoxy-(2S)-methylpentyl]-(4S)-Z-propenylpyrrolidine-(2R)-carboxylicacid, HCl salt) (0.031 mmol), cupric sulfate (0.62 mmol),tris(3-hydroxypropyltriazolylmethyl)-amine (0.62 mmol), and sodiumascorbate (0.93 mmol). The resulting homogeneous solution is gentlyshaken with a rocker table for 12 h. The crude solution is diluted withpH 7.4 PBS to a final concentration of 1 mg/mL, and ultrafiltered(10,000 MWCO) to a volume of 1 mL, for two times. The crude mixture isthen diluted 1:10 in PBS pH 7.4, and purified using MabSelect Sure Resin(GE Healthcare, Chicago, Ill., USA), followed by size exclusionchromatography. Purified material is quantified using a Nanodrop™ UVvisible spectrophotomer (using a calculated extinction coefficient basedon the amino acid sequence of the Fc used in the conjugation, andconcentrated to approximately 10 mg/mL using a centrifugal concentrator(10,000 MWCO). Purified molecules are analyzed using 4-12% Bis Tris SDSPAGE gels by loading 1-2 ug of each molecule into the gel, and stainingusing instant Blue staining. Each gel includes a molecular weight ladderwith the indicated molecular weight standards. MALDI MS analysis is usedto determine the average DAR.

Example 54. Synthesis of Int-15, HCl Salt

Step a.N-{(2.S)-methoxy-((1R)-[1-(4-methoxybenzyl)-5-oxo-(3S)-Z-propenylpyrrolidin-(2R)-yl]-(2S)-methylpentyl}-(3-butynyl)-carboxyamide

To 0° C. a stirring mixture of{(2S)-Methoxy-(1R)-[1-(4-methoxybenzyl)-5-oxo-(3S)-Z-propenylpyrrolidin-(2R)-yl]-(2S)-methylpentyl}-carbamicacid tert-butyl ester (it is prepared accordingly to reference JACS,2002, 124, 4716-4721; 1.0 mmol) in dry dichloromethane (5 mL) is addedtrifluoroacetic acid (10 mmol), and the temperature is raised toambient. Upon completion, the solvent is removed under reduced pressure.The resulting residue is dissolved in dichloromethane (20 mL) andextracted with a saturated aqueous solution of sodium bicarbonate. Theorganic layer is separated, dried (sodium sulfate), filtered andevaporated. The crude amine is dissolved in DMF (5 mL) and treated at 0°C. under stirring with 4-pentynoic acid (1.1 mmol),diisopropylethylamine (3.0 mmol) and HATU (1.1 mmol). Upon reactioncompletion, all the volatiles are evaporated per vacuum techniques. Theresidue is taken up in ethyl acetate (15 ml), and washed with saturatedaqueous solution of sodium bicarbonate (10 mL), then 1 M sulfuric acidaqueous solution (10 mL). The combined organics are dried over sodiumsulfate, filtered and concentrated. The residue is purified bychromatographic techniques to afford the desired product.

Step b.(2R)-((1R)-(4-pentynoyl)-(2S)-methoxy-(2S)-methylpentyl)-(5R)-oxo-(3S)-Z-propenylpyrrolidine-1-carboxylicacid tert-butyl ester

To a stirring solution ofN-{(2.S)-methoxy-((1R)-[1-(4-methoxybenzyl)-5-oxo-(3S)-Z-propenylpyrrolidin-(2R)-yl]-(2S)-methylpentyl}-(3-butynyl)-carboxyamide(1.0 mmol) in a mixture of acetonitrile and water (10:1.5 mL) is addedceric ammonium nitrate (2.0 mmol) in small portions at 45° C. during 1h, and stirring is continued until completion. The reaction is quenchedwith a saturated aqueous solution of sodium bicarbonate (5 mL). Theaqueous layer is extracted with EtOAc (3×10 mL), the combined organiclayers are dried (sodium sulfate) and evaporated to give a crude whichis used for the next step without further purification. The material isdissolved in acetonitrile (5 mL), di-tert-butylcarbonate is added (1.5mmol) followed by triethylamine (2.0 mmol) and DMAP (catalytic). Uponcompletion, the reaction is quenched with a saturated solution ofammonium chloride (5 mL). The aqueous layer is extracted with EtOAc(3×10 mL), and the combined organic layers are dried (sodium sulfate).All the volatiles are removed per vacuum techniques. If necessary, theresidue is purified by chromatographic techniques to afford the desiredproduct.

Step c.(2R)-((1R)-(4-pentynoyl)-(2S)-methoxy-(2S)-methylpentyl)-(5R/S)-methoxy-(3S)-Z-propenylpyrrolidine-1-carboxylicacid tert-butyl ester

To a −78° C. stirring solution of(2R)-((1R)-(2R)-((1R)-(4-pentynoyl)-(2S)-methoxy-(2S)-methylpentyl)-(5R)-oxo-(3S)-Z-propenylpyrrolidine-1-carboxylicacid tert-butyl ester (1.0 mmol) in THF (8 mL) is added SUPER-HYDRIDE®(1 M in THF, 2.2 mmol). After 30 min the reaction mixture is quenchedwith a saturated aqueous solution of sodium bicarbonate (4 mL) and 30%hydrogen peroxyde (5 drops). The mixture is warmed up to rt and stirredfor another 30 min and the aqueous layer is extracted with EtOAc (3×10mL). The combined organic layers are dried (sodium sulfate) and thesolvent is evaporated to give the hemiaminal, which is used withoutfurther purification. To a solution of the above product in methanol (16mL) is added p-toluenesulfonic acid hydrate (0.1 mmol) at rt. Thereaction mixture is stirred overnight and is quenched with a saturatedaqueous solution of sodium bicarbonate (10 mL). Methanol is removedunder reduced pressure, water (10 mL) is added to the resulting residueand extracted with EtOAc (3×10 mL). The organics are separated and driedwith brine and sodium sulfate, filtered and concentrated. If necessary,the residue is purified by chromatographic techniques to afford thedesired product.

Step d.(2R)-((1R)-(4-pentynoyl)-(2S)-methoxy-(2S)-methylpentyl)-(5R)-cyano-(3S)-Z-propenylpyrrolidine-1-carboxylicacid tert-butyl ester

To a −78° C. stirring solution of(2R)-((1R)-(4-pentynoyl)-(2S)-methoxy-(2S)-methylpentyl)-(5R/S)-methoxy-(3S)-Z-propenylpyrrolidine-1-carboxylicacid tert-butyl ester (1.0 mmol) in dichloromethane (20 mL) it is addedtrimethylsilyl cyanide (2.0 mmol) followed by boron trifluoride diethyletherate (1.2 mmol). The reaction mixture is stirred from −78° C. to−50° C. over a period of 3 h. A saturated aqueous solution of sodiumbicarbonate (40 mL) is added and the aqueous layer is extracted withEtOAc (3×15 mL). The combined organic layers are dried (sodium sulfate),filtered and concentrated under reduced pressure. The resulting residueconsisting of a mixture of epimeric cyano derivatives is purified bychromatographic techniques to afford the desired product.

Step e

To a stirring mixture ofN-{(2.S)-methoxy-((1R)-[1-(4-methoxybenzyl)-5-oxo-(3S)-Z-propenylpyrrolidin-(2R)-yl]-(2S)-methylpentyl}-(3-butynyl)-carboxyamide(1.0 mmol), bis-[N′-(2-azidoethyl)]-iminodiacetic amide (0.5 mmol),1H-1,2,3-Triazole-4-methanamine,1-(phenylmethyl)-N,N-bis[1-(phenylmethyl)-1H-1,2,3-triazol-4-yl]methyl]-(0.1 mmol) and sodium ascorbate (1.0 mmol) in ethanol (5 mL) and water(5 mL), it is added copper sulfate (0.1 mmol). Upon completion, thereaction is treated with SiliaMetS TAAcONa (0.3 mmol) for 30 minutes.The reaction is filtered and all the volatiles are evaporated per vacuumtechniques. The residue is purified by chromatographic techniques toafford the desired product.

Step f

To 0° C. a stirring solution of the intermediate from step e. (1.0mmol), propargyl-PEG4-acid (1.05 mmol), and DIPEA (3.0 mmol) in DMF (10mL) it is added HATU (2.0 mmol). All the volatiles are evaporated pervacuum techniques and the residue is purified by chromatographictechniques to afford the desired product.

Step g. Int-15, HCl Salt

To a solution of the intermediate from step f. (1.0 mmol) in AcOH (10mL), 12N HCl (10 mL) is added at rt. The solution is stirred at rt untilcompletion, the solvent is evaporated under reduced pressure. Ifnecessary, the residue is purified by chromatographic techniques toafford the desired product.

Example 55. Synthesis of Conjugate 11

A solution of hIgG1 Fc-PEG4-azide in pH 7.4 PBS×1 buffer solution (100mg, 1.71 umol, 7.011 mL, MW=58,200 Da, DAR=3.7) is added to a pH 7.4PBS×1 buffer solution (2.45 mL) of Int-15 HCl salt (0.031 mmol), cupricsulfate (0.62 mmol), tris(3-hydroxypropyltriazolylmethyl)-amine (0.62mmol), and sodium ascorbate (0.93 mmol). The resulting homogeneoussolution is gently shaken with a rocker table for 12 h. The crudesolution is diluted with pH 7.4 PBS to a final concentration of 1 mg/mL,and ultrafiltered (10,000 MWCO) to a volume of 1 mL, for two times. Thecrude mixture is then diluted 1:10 in PBS pH 7.4, and purified usingMabSelect Sure Resin (GE Healthcare, Chicago, Ill., USA), followed bysize exclusion chromatography. Purified material is quantified using aNanodrop™ UV visible spectrophotomer (using a calculated extinctioncoefficient based on the amino acid sequence of the Fc used in theconjugation, and concentrated to approximately 10 mg/mL using acentrifugal concentrator (10,000 MWCO). Purified molecules are analyzedusing 4-12% Bis Tris SDS PAGE gels by loading 1-2 ug of each moleculeinto the gel, and staining using instant Blue staining. Each gelincludes a molecular weight ladder with the indicated molecular weightstandards. MALDI MS analysis is used to determine the average DAR.

Example 56. Synthesis of bis-[N′-(2-azidoethyl)]-iminodiacetic amide

Step a. Bis-[N′-(2-azidoethyl)]-N-Boc-iminodiacetic amide

To 0° C. a stirring solution of N-Boc-iminodiacetic acid (1.0 mmol),2-azidoethan-1-amine hydrochloride (2.0 mmol), and DIPEA (6.0 mmol) inDMF (10 mL) it is added HATU (2.0 mmol). All the volatiles areevaporated per vacuum techniques and the residue is purified bychromatographic techniques to afford the desired product.

Step b. Bis-[N′-(2-azidoethyl)]-iminodiacetic amide

To a 0° C. stirring mixture ofBis-[N′-(2-azidoethyl)]-N-Boc-iminodiacetic amide (1.0 mmol) and2-methyl-2-butene (0.5 mL) in dichloromethane (8.0 mL), it is addedtrifluoroacetic acid (4.0 mL). After 10 minutes, the temperature israised to ambient. Upon completion, all the volatiles are evaporated pervacuum techniques. The residue is purified by chromatographic techniquesto afford the desired product.

Example 57. Synthesis of Int-16

A monomer of sulfozanamivir conjugated to a PEG₄-alkyne linker and whichmay be further conjugated to an Fc domain or an albumin protein isproduced according to the following synthetic scheme. Sulfozanamivirstarting material is produced according to Hadhazi et al. Asulfozanamivir analogue has potent anti-influenza virus activity.ChemMedChem Comm. 13:785-789 (2018).

Example 58. Synthesis of Int-17

A dimer of sulfozanamivir conjugated to a PEG₄-alkyne linker and whichmay be further conjugated to an Fc domain or an albumin protein isproduced according to the following synthetic scheme. Sulfozanamivirstarting material is produced according to Hadhazi et al. Asulfozanamivir analogue has potent anti-influenza virus activity.ChemMedChem Comm. 13:785-789 (2018).

Example 59. Synthesis of Conjugate 12

A solution of azido functionalized aglycosylated Fc (70 mg, 4.7 mL,1.3709 μmol) was added to a 15 mL centrifuge tube containing alkynederivatized small molecule (29.8 mg, 0.0216 mmol, Int-7). After gentlyshaking to dissolve all solids, the mixture was added with 206 μl of amixture solution of L-ascorbic acid sodium (59.4 mg, 0.3 mmol), copper(II) sulfate (15.9 mg, 0.1 mmol), and THPTA (43.5 mg, 0.1 mmol) in PBS7.4 buffer (1 ml). The resulting mixture was gently shaken overnight. Itwas purified by affinity chromatography over a protein A column,followed by size exclusion chromatography as described in Example 8.Maldi TOF analysis of the purified final product gave an average mass of56,177 Da (DAR=3.6). Yield 10.1 mg, 14% yield. A non-reducing SDS-PAGEof Conjugate 12 is provided in FIG. 54.

Example 60. Synthesis of Int-18

Step a. Synthesis of tert-butyl(17-{4-[(tert-butoxycarbonyl)amino]butyl}-16-oxo-4,7,10,13-tetraoxa-17-azahenicos-1-yn-21-yl)carbamate

To the solution of di-tert-butyl[azanediyldi(butane-4,1-diyl)]biscarbamate (1.5 g, 4.17 mmol fromExample 45, step a) and propargyl-PEG4-acid (1.08 g, 4.17 mmol) in DCM(40 ml) was added EDC (1.0 g, 5 mmol), HOBt (650 mg, 5 mmol), and DIEA(1.4 ml, 10 mmol) at room temperature, then stirred overnight at roomtemperature. The resulting solution was concentrated and purified by andpurified by reverse phase liquid chromatography (RPLC) using an IscoCOMBIFLASH® liquid chromatograph eluted with 10% to 100% acetonitrileand water with no modifier. Yield of 1.9 g, 76%. Ion(s) found by LCMS:M+H=602.4.

Step b. Synthesis ofN,N-bis(4-aminobutyl)-4,7,10,13-tetraoxahexadec-15-yn-1-amide

Tert-butyl(17-{4-[(tert-butoxycarbonyl)amino]butyl}-16-oxo-4,7,10,13-tetraoxa-17-azahenicos-1-yn-21-yl)carbamate(1.90, 3.1 mmol) was treated with 20 ml TFA at room temperature for 0.5hour, then concentrated to dryness and used in the next step withoutfurther purification. Yield is quantitative for this step, Ion(s) foundby LCMS: M/2+H=402.3.

Step c. Synthesis of Full Protected Int-18

N,N-bis(4-aminobutyl)-4,7,10,13-tetraoxahexadec-15-yn-1-amide (0.150 g,0.32 mmol) was added to a solution of the ether linked of zanamivir acid(0.400 g, 0.63 mmol, described in Example 31, step f) in DCM (10 mL),then treated with EDC (0.200 g, 1.0 mmol), HOBt (0.135 g, 1.00 mmol),and DIEA (0.14 ml, 1.00 mmol) at room temperature for overnight. Theresulting solution was concentrated and purified by and purified byreverse phase liquid chromatography (RPLC) using an Isco COMBIFLASH®liquid chromatograph eluted with 10% to 100% acetonitrile and water withno 0.1% TFA as modifier. Yield of the products 310 mg, 60.3%. Ion(s)found by LCMS: M/2+H=813.9.

Step d. Synthesis Int-18

Product from the previous step (300 mg, 0.18 mmol) was treated withtrifluoroacetic acid (2 mL) acid and stirred for 30 min at roomtemperature. The resulting solution was concentrated, re-dissolved inwater (2 mL), then treated with a solution of lithium hydroxide (24 mg,1 mmol) dissolved in water (1 mL). The reaction was stirred 10 min thenquenched with 0.1 ml acetic acid, concentrated and purified by reversephase liquid chromatography (RPLC) using an Isco COMBIFLASH® liquidchromatograph eluted with 0% to 50% acetonitrile and water, using 0.1%TFA as the modifier. Yield of the products 140 mg, 52%. Ion(s) found byLCMS: M/2+H=573.8, M/3+H=382.9.

Example 61. Synthesis of PEG4-Azido Fc for Conjugate 13a

c-Preparation of 0.05M PEG4-azidoNHS Ester Solution in DMF/PBS

PEG4-azido NHS ester (80.5 mg) was dissolved in DMF (0.50 mL) at 0° C.and diluted to 4.063 mL by adding 3.50 mL of PBS 1× buffer at 0° C. Thissolution was used for preparing other PEG4-azido Fc with variety ofdrug-antibody ratio (DAR) values by adjusting the equivalents of thisPEG4-azido NHS ester PBS×1 solution.

Preparation of PEG4-Azido Fc

0.05M PEG4-azidoNHS ester PBS×1 buffer solution (0.0984 mL, 4.92 μmol,2.5 equivalents) was added to a solution of h-IgG1 Fc (105 mg in 5.031mL of pH 7.4 PBS, MW˜53,360 Da, 1.968 μmol) and the mixture was shakengently for 12 hours at ambient temperature. The solution wasconcentrated using a centrifugal concentrator (30,000 MWCO) to a volumeof 1.5 mL. The crude mixture was diluted 1:10 in PBS pH 7.4, andconcentrated again. This wash procedure was repeated for total of threetimes. The unreacted azido reagent was removed with this procedure. Theconcentrated Fc-PEG4-azide was diluted to 5.03 mL with pH 7.4 PBS 1×buffer and ready for click conjugation. The purified material wasquantified using a Nanodrop™ UV visible spectrophotomer (using acalculated extinction coefficient based on the amino acid sequence ofh-IgG1). The yield was quantitative after buffer exchange/purification.

Example 62. Synthesis of Conjugate 13a

Preparation of click reagent solution: 0.0050M CuSO₄ in PBS buffersolution: 20.0 mg CuSO₄ was dissolved in 25.0 mL PBS×1, than took 22.0mL above CuSO₄ solution and added 189.4 mg BTTAA and 1090 mg NaAscorbate to give a clear solution (0.0050M CuSO4, 0.020M BTTAA and0.25M Sodium Ascorbate).

A solution of azido functionalized Fc (100 mg, 4.79 mL, 1.87 μmol,CTP-161-4A-Linker-1-Azide) was added to a 15 mL centrifuge tubecontaining alkyne derivatized small molecule, Int-18 (11.2 mg, 0.00750mmol, prepared in Example 60). After gently shaking to dissolve allsolids, the solution was treated with 3.00 mL of above click reagentsolution. The resulting colorless homogeneous solution was gently shakenovernight. It was purified by affinity chromatography over a protein Acolumn, followed by size exclusion chromatography (see general conjugatepurification protocol in Example 10). Maldi TOF analysis of the purifiedfinal product gave an average mass of 55,913 Da (DAR=1.7). Yield 54.0mg, 54% yield.

Example 63. Syntheses of Conjugate 13b, Conjugate 13c, Conjugate 13d,Conjugate 13e, Conjugate 13f, and Conjugate 13g

The PEG4-azido Fes for Conjugate 13b, Conjugate 13c, Conjugate 13d,Conjugate 13e, Conjugate 13f, and Conjugate 13g were preparedanalogously to the PEG4-azido Fc of Conjugate 13a (Example 61) adjustingthe number of equivalents of PEG4-azido NHS ester as described in thetable below. Conjugate 13b, Conjugate 13c, Conjugate 13d, Conjugate 13e,Conjugate 13f, and Conjugate 13g were prepared analogously to Conjugate13a in Example 62 where the number of equivalents of targeting moiety(Int-18) was adjusted based on the desired DAR value (Table 26), and thevolume of click reagent solution used was the same volume as employed inthe procedure for Example 62. The DAR values, molecular weights andyields are listed in the table below. Product conjugates were purifiedby affinity chromatography over a protein A column, followed by sizeexclusion chromatography as described in Example 8. A non-reducingSDS-PAGE of Conjugates 13a-13g is provided in FIG. 55.

TABLE 26 Yields of Conjugates 13a-13g Equivalents of PEG4- MALDI Sampleazido NHS DAR mass Da Targeting Equivalents Protein Name ester (Average)(Average) moiety (TM) of TM Type Yield (%) Conjugate 13a 2.5 1.7 55913Int-18 4 hlgG1 Fc 54% Conjugate 13b 4.5 2.7 57278 Int-18 6 hlgG1 Fc 50%Conjugate 13c 6.5 3.8 58908 Int-18 8 hlgG1 Fc 60% Conjugate 13d 8.5 4.760149 Int-18 10 hlgG1 Fc 54% Conjugate 13e 11 5.8 61726 Int-18 12 hlgG1Fc 55% Conjugate 13f 18 8.2 65146 Int-18 16 hlgG1 Fc 53% Conjugate 13g25 10.3 68153 Int-18 20 hlgG1 Fc 45%

Example 64. In Vitro Stability of Conjugate 6

To demonstrate the in vitro stability of Conjugate 6 using both mouseand human fresh K₂EDTA treated plasma and liver microsomes. The in vitromouse and human plasma stability was determined by comparing the Drug toAntibody Ratio (DAR) envelope after a 24 hr incubation in plasma at 37°C. by MALDI-TOF mass spectrometric detection. Liver microsomal stabilityusing mouse and human microsomes was also performed after incubation for24 hr at 37° C. with MALDI-TOF mass spectrometric detection. This was toidentify potential metabolically labile sites on the Fc protein, linker,or target moiety.

64.1 Plasma Stability Sample Preparation

First, 60 μL of Conjugate 6 at 3 mg/ml was mixed with 120 μL plasma.Each plasma type was aliquoted into 2 tubes. One aliquot was immediatelyfrozen from each plasma type. The remaining aliqoute was placed in awater bath (37° C.) for 24 hours. MAGNE® Protien A beads (Promega) wereequilibrated by gently vortexing the beads into suspension. In duplicatefor both plasma types, 50 μL of bead slutty was added to a 1.5 mLmicrocentrifuge tube and placed on the magnetic stand for 10 seconds.After 10 seconds, the storage buffer was removed and discarded. 500 μLof bind/wash buffer (0.1% BSA in 1×PBS pH 7.4) was added to the 1.5 mLmicrocentrifuge tube containing the beads. The beads were mixed(vortexing) and placed on a magnetic stand for 10 seconds. After 10seconds, the bind/wash buffer was removed and discarded. 50 μL of buffer(1×PBS, pH 7.4) was added to the microcentrifuge tube containing thebeads. 50 μL of the plasma mixture was added to the beads and gentlyvortexed to mix. Using a tube shaker, the sample was mixed at roomtemperature for 60 minutes, ensuring the beads remained in suspension.After mixing, the tube was placed on a magnetic stand for 10 seconds andthe supernatant was removed. 500 μL of buffer (1×PBS, pH 7.4) was addedand gently vortexed to mix. After mixing, the tube was placed on amagnetic stand for 10 seconds, followed by removal and discarding ofwash buffer. The wash step was repeated for a total of 2 washed.Following 2 washes with 500 μL of buffer (1×PBS, pH 7.4), 3 washes with500 μL, 200 μL, and 100 μL, respectively, of water were performed. Theappropriate volume of water was added to the tube and gently vortexed tomix well and then placed on a magenetic stand for 10 seconds prior toremoval and discarding of the water. After the third wash with water, 30μL of elution buffer (90:10:0.4 Water:Acetonitrile:TFA) was added to thebeads. Using a tube shaker, the elution buffer and sample were mixed for30 minutes at room temperature. After mixing, the tube was placed on amagnetic stand for 10 seconds, the elution buffer, containing thesample, was removed and kept. 2 μL of sample were mixed with 2 μL ofMALDI matrix (20 mg/mL Sinapic Acid in 70:30:0.1 water:acetonitrile:TFA)and spotted onto a MALDI target plate using a dual layer technique. Thesample was then analyzed by MALDI-TOF mass spectrometry.

64.2 Liver Microsomal Sample Preparation

A 10× buffer was made with 500 mM Tris-HCl at pH 7.5 and 50 mM magnesiumchloride hexahydrate. ACV-006 was diluted to 50 μM in 1×PBS, pH 7.4.Liver microsomes were thawed and vortexed. An aliquot of each species ofliver microsomes (human and mouse) were heat killed at 70° C. for 15minutes for use as a control. Reaction mixtures were prepared for bothspecies according to Table 27. Tubes were incubated in a water bath (37°C.) for 24 hours. Samples were extracted for analysis using MAGNE®Protein A beads (Promega) following the protocol from 59.1.

TABLE 27 0.5 mg/mL final microsomes concentration with 5 μM Conjugate 6Heat Killed Live Total 400 μL 400 μL Water 286 μL 286 μL 10× Buffer  40μL  40 μL Microsomes (20 mg/mL)  10 μL  10 μL Compound (50 μM)  40 μL 40 μL NADPH Regenerating Solution A  20 μL  20 μL NADPH RegeneratingSolution B  4 μL  4 μL

64.3 Sample and Data Analysis

Samples were acquired using Bruker Compass Flex Control version 3.4 toobtain full scan MALDI-TOF mass spectra (Table 28). BSA was used as aninternal calibrant for the acquisition mass range. Data was furtheranalyzed with Bruker Compass Flex Analysis version 3.4 software. Inaddition, the DAR pattern of the control is compared to the DAR patternof the test sample.

TABLE 28 Mass Spectrometer (MS) Parameters Mass Spectrometer BrukerMicroflex LT Detection Mass Range 17-141 kDa Sample Rate and DigitizerSettings 0.5 Detector Gain 2.05× Baseline Offset Adjustment  0% AnalogOffset    0.5 mV Laser Frequency   60 Hz Spectrometer Ion Source 1 (IS1)20.03 kV Ion Source 2 (IS2) 18.12 kV (90.5% IS1) Lens  7.17 kV (35.8%IS1) Pulsed Ion Extraction  1010 ns Sample Carrier Random Walk PartialSample Laser Shots 100/section Total Laser Shots 500 Shots at Raster 20Raster Shot Diameter Limit 2000 um Setup Laser Global Attenuator Offset 0% Laser Attenuator Offset 20% Laser Attenuator Range 30% DigitizerSensitivity (Full Scale)   100 mV Digitizer Analog Offset Linear    0.5mV Digitizer Digital Offset Linear 0 cnt Detector Gain Voltage - LinearBase  2500 V Detector Gain Voltage - Linear Boost    0 V CalibrationBovine Serum Albumin [M + 2H], [M + 2H], [2M + H]

64.4 Results

The test compound, Conjugate 6 (FIG. 43), was tested for in vitrostability in both mouse and human fresh K₂EDTA treated plasma and livermicrosomes.

Conjugate 6 was spiked into fresh K₂EDTA mouse and human plasma at afinal concentration of 1 mg/mL. The plasma was split into 2 aliquots,one being frozen immediately, and the other incubated in a water bath at37° C. for 24 hours. At the end of the incubation, samples wereextracted from the plasma matrix by MAGNE® Protein A magnetic beads.Following plasma incubations, samples were analyzed by MALDI-TOF massspectrometry for changes in DAR. Conjugate 6 in either mouse (FIG. 44)or human (FIG. 45) plasma incubations were not found to generate anychanges in DAR.

Conjugate 6 liver microsomal stability was tested at a finalconcentration of 5 μM into a 50 mM, pH 7.5 Tris-HCl buffer solution thatcontained either active or heat killed liver microsomes at a finalconcentration of 0.5 mg/mL and MgCl₂ at a final concentration of 5 mM.All samples were incubated at a constant temperature of 37° C., andnicotinamide adenine dinucleotide phosphate (NADPH) regeneratingsolution was utilized to provide continuous cofactor availability duringthe incubation. Incubations were carried out for 24 hours. At the end ofthe incubation, samples were extracted from the microsomal matrix byProtein A magnetic beads. Following liver microsomal incubations,samples were analyzed by MALDI-TOF mass spectrometry for changes in DAR.Conjugate 6 in either mouse (FIG. 46) or human (FIG. 47) livermicrosomal incubations were not found to generate any changes in DAR.

The in vitro plasma stability after incubation at 37° C. for 24 hr,suggests a lack of degradation of the Conjugate 6 Fc, linker, ortargeting moiety in either mouse or human. Similarly a lack ofdegradation was observed after incubation in both mouse and human livermicrosomes, suggesting the absence of metabolites. The results of thesein vitro stability studies support that this is a stable compound withdegradants that could have biological liabilities.

Example 65. Efficacy of Conjugate 13 at Different Drug-to-AntibodyRatios (DARs) Against Influenza A (H1N1) in a Lethal Mouse Model

Conjugate 13 was evaluated against a lethal IAV H1N1 influenza infectionin female BALB/c mice (Charles River Laboratories, 6-8 weeks). Thechallenge virus (A/Puerto Rico/8/1934) is a mouse-adapted isolatecapable of causing lethal infections in mice. The experiment comprised13 groups of 5 mice. At day 0, all mice were challenged with virus at 3×the LD95 by intranasal inoculation in a volume of 30 μl, after beinganesthetized with a mixture of ketamine/xylazine (150 and 10 mg/kgrespectively).

Groups 1-13 received a single IV treatment, 2 hours post viral challengeof test article or vehicle (PBS). The study evaluated 4 different DARconstructs of Conjugate 13 corresponding to Conjugate 13a, Conjugate13c, Conjugate 13d, and Conjugate 13g (DARs of 1.7, 3.8, 5.8, and 10.3,respectively). The synthesis of Conjugate 13a, Conjugate 13c, Conjugate13d, and Conjugate 13g is described in Example 61-Example 63. Eachconstruct was evaluated at 0.03, 0.1, and 0.3 mg/kg. The general studydesign for each conjugate is summarized in Table 29.

TABLE 29 General study design of DAR scan Dose Route/ Dose volume GroupConjugate DAR Schedule (mg/kg) (ml/kg) 1 Conjugate 13a 1.7 IV, T + 2hours 0.3 5 2 Conjugate 13a 1.7 IV, T + 2 hours 0.1 5 3 Conjugate 13a1.7 IV, T + 2 hours 0.03 5 4 Conjugate 13c 3.8 IV, T + 2 hours 0.3 5 5Conjugate 13c 3.8 IV, T + 2 hours 0.1 5 6 Conjugate 13c 3.8 IV, T + 2hours 0.03 5 7 Conjugate 13d 5.8 IV, T + 2 hours 0.3 5 8 Conjugate 13d5.8 IV, T + 2 hours 0.1 5 9 Conjugate 13d 5.8 IV, T + 2 hours 0.03 5 10Conjugate 13g 10.3 IV, T + 2 hours 0.3 5 11 Conjugate 13g 10.3 IV, T + 2hours 0.1 5 12 Conjugate 13g 10.3 IV, T + 2 hours 0.03 5 13 Vehicle(PBS) Na IV, T + 2 hours na 5

All constructs were fully protective at 0.3 mg/kg, in contrast, noconstruct was active at 0.03 mg/kg (0% survival for all groups)indicating the low dose was below the threshold efficacious dose.However, groups receiving 0.1 mg/kg of conjugates could be discriminated(Table 30). At this dose level conjugates with DARs of 1.7, 3.8, and 5.8were significantly more protective than vehicle only treated mice(p=0.0027). The high DAR construct (10.3) however was not significantlymore protective than vehicle only treated mice (p=0.091). The underlyingmechanism by which the high DAR construct loses activity is currentlyunknown but could be caused by several factors, including interferencewith antibody recycling, resulting in shorter half-life.

TABLE 30 DAR Range Study (0.1 mg/kg dose groups) Conjugate DAR %Survival Significance* Conjugate 13a 1.7 60 p = 0.0027 Conjugate 13c 3.840 p = 0.0027 Conjugate 13d 5.8 80 p = 0.0027 Conjugate 13g 10.3 0 p =0.091** *= Significance relative to vehicle (PBS) only treated mice bythe Log-rank (Mantel-Cox) test. **= Not significant

Example 66. In Vivo Efficacy of Conjugate 6 and Conjugate 12

Conjugate 6 and Conjugate 12, analog with Fc mutation at N297A, wasevaluated against a lethal IAV H1N1 influenza infection in female BALB/cmice (Charles River Laboratories, 6-8 weeks). The challenge virus(A/Puerto Rico/8/34) is a mouse-adapted isolate. The experimentcomprised 5 mice per group. Mice were anesthetized withketamine/Xylazine (150/10 mg/kg) and were challenged with influenzavirus at 3-5× the LD₉₅ by intranasal inoculation in a volume of 30 μL. Asingle 1 dose of treatment was administered by IV, 2 h post-infection.PBS was given as negative control. All mice were monitored for % bodyweight loss (FIG. 48) and for survival (FIG. 49) for 15 days afterchallenge.

Example 67. In Vitro Fcγ Receptor IIIA Binding of Conjugate 6 andConjugate 12

Binding of Conjugate 6 and Conjugate 12, analog with Fc mutation atN297A, was evaluated against FcγRIIIA by ELISA. The plate was coatedwith 1 μg/mL recombinant human FcγRIIIA overnight. The next day, theplate was blocked with 1% BSA solution for 1 h. Conjugates were added toplate in dose-response ranging from 0.01-1000 nM and incubated for 2 h.Binding was detected by incubation with peroxidase-conjugated anti-humanFc for 1 h and subsequent incubation with TMB substrate reagent for10-15 min.

Binding was determined by reading absorbance at 450 nm (FIG. 50 and FIG.51).

Example 68. In Vivo Conjugate 6 Plasma Sample Analysis

Conjugate 6 in plasma samples were quantified by a neuraminidase capturedetection ELISA. Briefly, molecules were captured on neuraminidasecoated plates and then detected using a HRP-conjugated anti-human IgG-Fcantibody. Protein concentration was calculated in GraphPad Prism using4PL non-linear regression of Conjugate 6 standard curves. A moredetailed method description is provided below.

Nunc Maxisorp 96-well plates (Cat No. 12-565-136, ThermoFisher) werecoated with 0.1 U/well neuraminidase from A/California/04/2009 (H1N1)(11058-VNAHC, Sino Biological) in 1×KPL coating buffer (5150-0041,SeraCare). Plates were incubated at room temperature for 1 hr on anorbital plate shaker (500 rpm). Serial dilutions of the plasma sampleswere plated and incubated at room temperature for 2 hours (samplediluent: 0.5% BSA in PBS 0.025% Tween 20+naïve cynomolgus monkey plasmafinal concentration of 1:2,500). Conjugate 6 standard curves rangingfrom 0.230 to 500 ng/mL, in duplicate were run on each plate. Followingthe 2 hr incubation, plates were washed 5× in 300 μL PBS with 0.05%Tween 20. Conjugate bound to neuraminidase on the plates was then probedwith an HRP conjugated anti-human IgG Fc F(ab′)2 (709-036-098, Jackson)diluted 1:1,000 in sample diluent for 1 hr at room temp. Plates werethen washed 8× in 300 uL PBS with 0.05% Tween 20 and developed with TMBsubstrate for 7-8 minutes. The reaction was stopped with 1N H₂SO₄.Absorbance was read at 450 nm. Conjugate 6 in plasma samples wasinterpolated using GraphPad Prism Version 6 following nonlinearregression analysis (Sigmoidal, 4PL analysis) of the standard curves.

The resulting mean plasma concentrations were then used to calculatepharmacokinetic parameters by non-compartmental analysis using PhoenixWinNonlin 7.0.

Toxicokinetics (TK), Groups 2 (IV, 5 mg/kg) and 3 (IV, 20 mg/kg)

Concentrations were comparable between male and female animals withinthe same dose group on day 1 (Table 31) and day 8 (Table 32) afteradministration. Mean plasma exposures appeared to increase approximatelydose-proportionally across both days. After the 2nd dose administration,a slight accumulation of about 30% was noted across the different dosegroups. A plot of days 1 and 8 mean plasma concentrations across thedifferent dose groups is shown in FIG. 52.

TABLE 31 Toxicokinetics Day 1 Dose Conc (μg/mL) at Time (hr) Tmax CmaxAUC0-t (mg/kg) Route Sex 0.083 1 2 4 8 24 72 120 168 (hr) (μg/mL) (μg ·hr/mL) 5 IV F 112 78.8 85.8 71.0 75.1 52.5 38.6 18.5 22.3 0.083 112 6190IV M 101 84.6 107 75.3 72.5 38.1 29.8 35.3 15.8 2 107 5970 Mean 107 81.796.5 73.2 73.8 45.3 34.2 26.9 19.0 1.04 110 6080 20 IV F 449 510 353 331303 134 105 63.6 46.6 1 510 18800 IV M 448 422 485 373 329 160 96.0 76.253.4 2 485 20500 Mean 449 466 419 352 316 147 100 69.9 50.0 1.50 49719600

TABLE 32 Toxicokinetics Day 8 Dose Conc (μg/mL) at Time (hr) Tmax CmaxAUC0-t (mg/kg) Route Sex 0.083 1 2 4 8 24 72 120 168 (hr) (μg/mL) (μg ·hr/mL) 5 IV F 111 111 99.3 82.3 100 52.2 46.7 35.0 33.9 1 111 7970 IV M113 114 113 78.4 74.4 54.7 35.2 47.1 28.2 1 114 7700 Mean 112 112 10680.4 87.3 53.5 41.0 41.1 31.0 1 112 7830 20 IV F 398 359 319 315 298 190126 99.3 85.5 0.083 398 23900 IV M 391 443 354 435 263 219 143 142 83.81 443 27800 Mean 394 401 337 375 280 205 134 120 84.7 0.542 420 25800Pharmacokinetics (PK), Groups 4 (IV, 10 mg/kg) and 5 (SC, 10 mg/kg)

Following IV administration, plasma concentrations from the male andfemale animals were comparable. Very low clearance, resulting in a longterminal half-life was observed following IV administration (Table 33Aand Table 33B).

TABLE 33A Plasma concentrations for male and female animals Dose Conc(μg/mL) at Time (hr) (mg/kg) Route Sex 0.083 1 2 4 8 24 72 120 168 336504 672 5 IV F 166 174 220 153 144 58.6 40.6 21.2 20.7 14.7 n/a 2.7 5 IVM 191 135 258 161 154 71 43.7 47.6 32 22.9 n/a 5.3

TABLE 33B PK parameters for male and female animals T_(max) C0 C_(max)AUClast AUCINF_obs Half-life Cl_obs Vss_obs Vz_obs (hr) (μg/mL) (μg/mL)(hr*μg/mL) (hr*μg/mL) (hr) (mL/min/kg) (mL/kg) (mL/kg) 1 166 220 1370014400 170 0.00579 68.9 85.1 1 204 258 19400 20800 183 0.004 56.6 63.2

Following SC administration, the time to reach maximum concentrationswas reached 72 hours after dosing but concentrations were measurablethrough 672 hours post-dose (Table 34A and Table 34B). Bioavailabilityafter SC dosing was high at approximately 139%. A comparison of theplasma concentration overtime between 10 mg/kg IV and SC administrationis shown in FIG. 53.

TABLE 34A Plasma concentrations for male and female animals Dose Conc(μg/mL) at Time (hr) Route (mg/kg) Sex 0.083 0.5 1 4 8 24 72 120 168 336504 672 SC 10 F n/a 1.9 5.3 23.1 39.5 54 58.6 66.9 54 28.4 18.9 6.4 SC10 M n/a 0.3 2.1 19.9 28.1 46.8 64.9 54.7 57.2 26.8 25.5 8.6

TABLE 34B PK parameters for male and female animals T_(max) C_(max)AUC_(0−t) F (hr) (μg/mL) (μg × hr/mL) (%) 120 66.9 22600 137% 72 64.923300 141%

Example 69. Efficacy of Conjugate 6 Against Influenza A/PuertoRico/8/1934 (H1N1) in a Lethal Mouse Model

Conjugate 6 was evaluated against a lethal IAV H1N1 influenza infectionin female BALB/c mice (Charles River Laboratories, 6-8 weeks). Thechallenge virus (A/Puerto Rico/8/1934) is a mouse-adapted isolatecapable of causing lethal infections in mice. The experiment comprised 7groups of 5 mice. At day 0, all mice were challenged with virus at 3×the LD₉₅ by intranasal inoculation in a volume of 30 μl, after beinganesthetized with a mixture of ketamine/xylazine (150 and 10 mg/kgrespectively). Mortality and body weights were recorded daily and anyanimal with a 20% loss of body weight was scored as a death. Test groupsreceived a single IV treatment, 2 hours post viral challenge ofconjugate 6, hIgG1 Fc control, or vehicle (PBS). Animals receivingoseltamivir were dosed orally, twice daily, for 5 days, starting 2 hoursafter viral challenge. The study design is summarized in Table 35.

TABLE 35 Study design for Influenza A/PR/8/34 (h1N1) study Dose Dose(mq/ volume # of Group Test article Route/Schedule kq) (ml/kq) mice 1Vehicle IV, T + 2 hrs. na 5 5 2 hlgG1 Fc IV, T + 2 hrs. 10 5 5 3Oseltamivir PO, bid × 5, T + 2 hrs. 20 5 5 4 Oseltamivir PO, bid × 5,T + 2 hrs. 5 5 5 5 Conjugate 6 IV, T + 2 hrs. 10 5 5 6 Conjugate 6 IV,T + 2 hrs. 2 5 5 7 Conjugate 6 IV, T + 2 hrs. 0.4 5 5

As expected, mice receiving vehicle or the hIgG1 Fc only succumbed tothe infection by day 6. Similarly, mice treated with oseltamivir at thelow dose (5 mg/kg; bid for 5 days) reached mortality by day 8 (Table36). However, mice receiving 20 mg/kg of oseltamivir with the samedosing schedule were fully protected (p=0.0027). In contrast to thatseen with oseltamivir, mice treated with conjugate 6 were fullyprotected at all dose levels (10, 2, and 0.4 mg/kg) from a single IVdose (p=0.0027).

TABLE 36 Survival for Influenza A/PR/8/34 (H1N1) study (Day 14) Testarticle Dose % Survival Significance* hlgG1 Fc 10 0 p = 0.85**Oseltamivir 20 100 p = 0.0027 Oseltamivir 5 0 p = 0.091** Conjugate 6 10100 p = 0.0027 Conjugate 6 2 100 p = 0.0027 Conjugate 6 0.4 100 p =0.0027 *= Significance relative to vehicle (PBS) only treated mice bythe Log-rank (Mantel-Cox) test. **= Not significant

The potency of conjugate 6 was further supported by daily body weightmeasurements. As expected, mice treated with vehicle or hIgG1 Fcdemonstrated a steady drop in body weight until it exceeded 20%, atwhich time they were scored as a death (Table 37). The group treatedwith oseltamivir at 5 mg/kg also displayed a consistent loss of weightuntil reaching mortality at day 8. Mice treated with oseltamivir at thehigh dose (20 mg/kg) showed a steady, but reduced loss of body weight,which reached 14% at day 8, before recovering.

In contrast to control and oseltamivir treated mice, those groupsreceiving conjugate 6 maintained healthy body weights throughout thestudy even at the lowest dose concentration (0.4 mg/kg) (Table 37). Thelargest transient loss of weight among conjugate 6 treated mice was only2% at day 14 in the 2 mg/kg dose group. By both survival and body weightmeasurements conjugate 6 demonstrated robust protection from InfluenzaA/Puerto Rico/8/1934 with a single IV dose as low as 0.4 mg/kg.

TABLE 37 Mouse body weight data (% BW relative to day 0). Average of 5mice; *data not included once the first animal reaches mortality withina group hIgG1 Fc Oseltamivir Conjugate 6 Day post Vehicle (mg/kg)(mg/kg) (mg/kg) challenge (PBS) 10 20 5 10 2 0.4 0 100 100 100 100 100100 100 1 99 99 102 99 103 102 101 2 102 102 103 96 104 102 103 3 96 96102 95 102 102 100 4 89 88 100 90 103 100 100 5 82 81 95 89 102 99 99 677 77 98 90 102 100 101 7 90 84 103 101 102 8 86 78 105 101 102 9 91 101102 102 10 95 102 101 103 11 97 102 101 102 12 97 101 99 102 13 97 103100 102 14 96 104 98 101

Example 70. Synthesis of Conjugate 14

The title conjugate was prepared analogously to Conjugate 13a (Example61) using PEG-azido-Fc (SEQ ID NO:35) and Int-7 (Example 19). Maldi TOFanalysis of the purified final product gave an average mass of 56,502.Da (DAR=2.1). Yield 43.4 mg, 43.4%.

Example 71. Synthesis of Conjugate 15

This conjugate was prepared analogously to Example 62 (Conjugate 13a) byPEG4-azido-Fc (SEQ ID NO: 35, Example 61) and Int-12 (Example 46). MaldiTOF analysis of the purified final product gave an average mass of56,528. Da (DAR=2.2). Yield 40.0 mg, 40.0%.

Example 72. Synthesis of Conjugate 16

This conjugate was prepared analogously to Example 62 (Conjugate 13a) byPEG4-azido-Fc (SEQ ID NO: 35, Example 61) and Int-10 (Example 31). MaldiTOF analysis of the purified final product gave an average mass of56,507. Da (DAR=2.1). Yield 41.7 mg, 42%.

Example 73. Synthesis of Int-19

Step a

To a solution of tert-butyl [2-(2-bromoethoxy)ethyl]carbamate (1.8 g,6.6 mmol) and propargyl-PEG4 amine (0.7 g, 3.0 mmol) in 30 ml DMF wasadded potassium carbonate (1.2 g, 9 mmol). The reaction was stirred at80° C. for 6 hrs, and then partitioned between DCM (200 mL) and brine(50 mL). The organic layer was separated and washed with brine and driedwith anhydrous sodium sulfate. Upon filtration, the resulting filtratewas concentrated and purified by reverse phase liquid chromatography(RPLC) using an Isco COMBIFLASH® liquid chromatograph eluted with 10% to100% acetonitrile and water with 0.1% TFA as modifier. Yield of theproducts 1.0 g, 65%. Ion(s) found by LCMS: M/2+H=606.4.

Step b

Product from the previous step (1.0 g, 1.6 mmol) was treated with TFA(10 mL) at room temperature for 0.5 hour, then concentrated to drynessand used in next step without further purification. Yield wasquantitative for this step, Ion(s) found by LCMS: M/2+H=406.3.

Step c

Product from the previous step (120 mg, 0.17 mmol) was treated with asolution of ether zanamivir acid (230 mg, 0.38 mmol, Example 31) in 10ml DMF. To this solution was added EDC (100 mg, 0.5 mmol), HOBt (65 mg,0.5 mmol), and DIEA (0.14 ml, 1 mmol). The resulting solution wasstirred at room temperature overnight, then purified by and purified byreverse phase liquid chromatography (RPLC) using an Isco COMBIFLASH®liquid chromatograph eluted with 10% to 100% acetonitrile and water withno TFA as modifier. Yield of the products 180 mg, 60.2%. Ion(s) found byLCMS: M/2+H=816.9.

Step d

Product from the previous step (180 mg, 0.11 mmol) was treated withtrifluoroacetic acid (2 mL) for 30 min at room temperature. Theresulting solution was concentrated and was dissolved into water (2 mL),then treated with a solution of lithium hydroxide (24 mg, 1 mmol)dissolved in H2O (1 mL). The resulting reaction was stirred 10 min thenquenched with 0.1 ml acetic acid. The solution was concentrated andpurified by reverse phase liquid chromatography (RPLC) using an IscoCOMBIFLASH® liquid chromatograph eluted with 0% to 50% acetonitrile andwater, using 0.1% TFA as the modifier. Yield of product 140 mg, 52%.Ion(s) found by LCMS: M/2+H=575.8, M/3+H=384.2.

Example 74. Synthesis of Conjugate 17

This conjugate was prepared analogously to Example 62 (Conjugate 13a) byPEG4-azido-Fc (SEQ ID NO: 35, Example 61) and Int-19 (Example 73). MaldiTOF analysis of the purified final product gave an average mass of56,672. Da (DAR=2.2). Yield 36.7 mg, 36.7%.

Example 75. Synthesis of Int-20

Step a

To a solution of diethyl iminodiacetate (3.1 g, 16.06 mmol) in anhydrousDMF (36 mL) was added benzyl bromide (2.38 mL, 19.64 mmol) and potassiumcarbonate (6.44 g, 46.46 mmol). The resulting mixture was heated at 70°C. for 16 hours. After cooling to room temperature, the reaction mixturewas diluted water and extracted with tertiarybutylmethylether (3×100mL). The organic layers were washed with brine, dried over Na₂SO₄ andfiltered then concentrated. The residue was purified by normal phasesilica gel chromatography (Isco, 0 to 10% ethyl acetate and hexane).Yield 3.13 g, 69.8%. Ion found by LCMS: [M+H]+=280.2.

Step b

A solution of di-ethyl ester from step-a (3.2 g, 11.2 mmol) in anhydrousTHF (5 mL) was added slowly to a round bottom flask containing LiALH₄(425 mg, 14.2 mmol) in THF (2 mL) under nitrogen gas at 0° C. Thesyringe was rinsed with THF (2×5 mL). The resulting mixture was slowlywarmed up to room temperature overnight. Methanol (2 mL) was addedslowly to quench the reaction following by addition of NaOH aqueous (1mL). The resulting mixture was stirred for 1 hour then filtered undervacuum. The filtrate was concentrated and used in the next step withoutpurification. Yield 2.4 g, 109%. Ion found by LCMS: [M+H]+=196.2.

Step c

Product from the previous step (1.02 g, 5.24 mmol) in anhydrous THF (4mL) was added slowly into a flask containing NaH (60% purity, 2.09 g,52.4 mmol) and THF (5 mL) at 0° C. under nitrogen gas. The resultingmixture was stirred for 1 hour followed by dropwise addition of3-(Boc-amino)propylbromide (3.8 g, 15.7 mmol) in THF (20 mL). Thereaction mixture was slowly warmed to room temperature and stirred for 3days. The reaction was cooled down to 0° C. then quenched with water (6mL) and stirred for 1 hour. It was extracted with ethyl acetate (2×100mL). The combined organic layers were washed with 1N HCl aqueous andbrine. It was dried over Na₂SO₄, filtered and concentrated. The residuewas purified by normal phase chromatography (Isco, 0 to 5% methanol anddichloromethane). Yield 984 mg, 37%. Ion found by LCMS [M+H]+=510.0.

Step d

Palladium hydroxide (543 mg, 0.77 mmol) was added into a flaskcontaining the step-c product (985.3 mg, 1.93 mmol) in anhydrousmethanol (19.5 mL), under an H₂ atmosphere. The resulting mixture wasstirred at room temperature for 16 hours. It was then filtered through apad of celite and washed with methanol, and concentrated. The residuewas carried on to the next step without purification. Yield 828.6 mg,102%. Ion found by LCMS [M+H]+=420.0.

Step e

The step-d product (829 mg, 1.97 mmol) in H₂O:THF (1:1, 16 mL) wascooled down to 0° C. To this solution was added Na₂CO₃ (314 mg, 2.96mmol) following by addition of Fmoc N-hydroxysuccinimide ester (826 mg,2.37 mmol). The resulting mixture was warmed up to room temperature andstirred until complete by LCMS, then extracted with ethyl acetate. Theorganic layer was washed with brine and dried over Na₂SO₄, filtered andconcentrated. The residue was purified by normal phase chromatography(Isco, 0 to 60% ethyl acetate and hexane). Yield 784 mg, 62%. Ion foundby LCMS [M+H-Boc]+=542.0.

Step f

The step-e product (1.01 g, 1.57 mmol) was stirred in TFA (5 mL) andCH₂Cl₂ (9 mL) at room temperature for 1 hour then concentrated underreduced pressure. The residue was purified by RPLP (Isco, 5 to 100%methanol and water without modifier). Yield 903 mg, 86%. Ion found byLCMS [M+H]+=442.2.

Step g

To a mixture of ether zanamivir acid (340 mg, 0.49 mmol), step-f product(111 mg, 0.25 mmol, Example 31) and HATU (206 mg, 0.53 mmol) inanhydrous DMF (3 mL) was added DIEA (162 mg, 1.23 mmol). The resultingmixture was stirred at room temperature for 1 hour, then directlypurified by RPLC (Isco, 30 to 100% methanol and water without modifier).Yield 249 mg, 61%. Ion found by LCMS [(M+2H)/2]+=833.8.

Step h

To a solution of the step-g product (249 mg, 0.15 mmol) in anhydrous DMF(0.5 mL) was added SilaMetS Thiol (1.2 g, 1.47 mmol) and1,8-diazabicyclo[5.4.0]undec-7-ene (12 mg, 0.07 mmol). The resultingmixture was stirred for 1.5 hours, then filtered directly into a by wayof containing HATU (69 mg, 0.18 mmol), propargyl PEG-4 acid (43 mg, 0.16mmol) and DIEA (43 mg, 0.33 mmol). The reaction mixture was stirred for1 hour, then directly purified by RPLC (Isco, 30 to 100% methanol andwater without modifier). Yield 298 mg, 118%. Ion found by LCMS[(M+2H-Boc)/2]+=843.9.

Step i

The step-h product (298 mg, 0.18 mmol) was dissolved in TFA (3 mL) andCH₂Cl₂ (3 mL), and the solution was stirred at room temperature for 16hours. It was then concentrated under reduced pressure and purified byHPLC (ACCQ Isco, 0 to 25% acetonitrile and water, using 0.1% TFA as amodifier). Yield 112 mg, 44%. Ions found by LCMS [(M+2H)/2]+=643.8 and[(M+3H)/3]+=429.6.

Step j

To a solution of the step-i product (112 mg, 0.072 mmol) in MeOH (4 mL)and water (2 mL) was added LiOH (10.6 mg, 0.43 mmol). The resultingsolution was stirred at room temperature for 1 hour, then acidified withTFA and concentrated under reduced pressure. The residue was purified byHPLC (ACCQ Isco, 0 to 25% acetonitrile and water, using 0.1% TFA asmodifier). Yield 37 mg, 36%. Ions found by LCMS [(M+2H)/2]+=603.8,[(M+3H)/3]+=402.9.

Example 76. Synthesis of Conjugate 18

A solution of azido functionalized aglycosylated Fc (100 mg, 5.4 mL,1.87 μmol, SEQ ID NO: 35) was added to a 15 mL centrifuge tubecontaining alkyne derivatized small molecule (16.1 mg, 0.011 mmol,Int-20). After gently shaking to dissolve all solids, the mixture wasadded to 3 mL premixed solution of L-ascorbic acid sodium (149 mg, 0.75mmol, 0.25 M), copper (II) sulfate (2.4 mg, 0.015 mmol, 0.005 M) andBTTAA (25.8 mg, 0.6 mmol, 0.02 M) in PBS 7.4 buffer. The resultingsolution was gently shaken overnight. It was purified by affinitychromatography over a protein A column, followed by size exclusionchromatography (see general conjugate purification protocol in Example10). Maldi TOF analysis of the purified final product gave an averagemass of 56826 Da (DAR 2.2). Yield 36.64 mg, 37%.

Example 77. Synthesis of Int-21

Step a

To a solution of 2-(2-Boc-aminoethoxy) ethanol (6.15 g, 30 mmol) inanhydrous DCM (60 ml) was added DIPEA (7.8 g, 60 mmol) and DMAP (366.6mg, 3 mmol). P-toluenesulfonyl chloride (6.86 g, 36 mmol) was then addedin portions over 30 minutes. After the resulting mixture was stirred for3 days, it was concentrated by rotary evaporation and purified by RPLC(20% to 70% acetonitrile/water with no modifier). Yield 3.71 g, 34.4%.Ion found by LCMS: [M-Boc+H]⁺=260.

Step b

To a solution of the step-a product (2.1 g, 5.83 mmol) in anhydrous THF(10 ml) was added sodium carbonate (1.24 g, 11.7 mmol) andmono-N-Boc-1,4-diaminobutane (1.32 g, 7 mmol). The resulting mixture washeated at 60° C. for 1 day. The salt was then filtered off, and thefiltrate was concentrated by rotary evaporation. The residue waspurified through RPLC (100 g, 5 to 50% acetonitrile and water, using0.1% TFA as modifier). Yield 1.94 g, 88.6%. Ion found by LCMS:[M+H]⁺=376.0.

Step c

To a solution of propargyl PEG-4 acid (781 mg, 3 mmol) and HATU (1.14 g,3 mmol) in anhydrous DMF (3 ml) was added DIPEA (390 mg, 3 mmol),followed by the addition of the solution step-b product (940 mg, 2.5mmol) and DIPEA (390 mg, 3 mmol) in anhydrous DMF (3 ml). The reactionmixture was stirred for 30 minutes, then directly purified through RPLC(100 g, 5 to 80% acetonitrile and water, using 0.1% TFA as modifier).Yield 960.2 mg, 65.3%. Ion found by LCMS: [M+H]⁺=618.3,[M-Boc+H]⁺=518.3.

Step d

The step-c product (960.2 mg, 1.63 mmol) was dissolved in anhydrous THF(6 ml). 4N HCl solution in dioxane (4 ml) was added, and the reactionmixture was stirred overnight. It was then concentrated by rotaryevaporation. The residue was extracted with water (3 ml×3) and ethylacetate (10 ml). The combined aqueous layers were lyophilized. Yield 760mg, 95.1%. Ion found by LCMS: [M+H]⁺=418.0.

Step e

To a mixture of ether zanamivir acid (315 mg, 0.5 mmol) and HATU (190mg, 0.5 mmol) in anhydrous DMF (1 ml) was added in portions a solutionof the step-d diamine product (148 mg, 0.3 mmol) and DIPEA (165 mg, 1.5mmol) in anhydrous DMF (1 ml) over 20 minutes. After the addition, thereaction was stirred for 30 more minutes and directly purified by RPLC(50 g, 30 to 90% acetonitrile and water, using 0.1% TFA as modifier).Yield 233 mg, 56.7%. Ion found by LCMS: [(M+2H)/2]⁺=821.3.

Step f

The step-e product (233 mg, 0.142 mmol) was dissolved in TFA (1.5 ml),and the solution was heated at 30° C. for 30 minutes. It was thenconcentrated and directly purified by RPLC (0% to 30% acetonitrile andwater, using 0.1% TFA as modifier). Yield 120 mg, 57.4%. Ions found byLCMS: [(M+2H)/2]⁺=621.4, [(M+3H)/3]⁺=414.7.

Step g

To a solution of the step-f product (120 mg, 0.0816 mmol) in MeOH (2 ml)was added LiOH monohydrate (63 mg, 1.5 mmol) solution in water (2 ml).The resulting mixture was stirred for 1.5 hours and then concentrated byrotary evaporation. The residue was acidified by 4N HCl solution indioxane (0.5 ml) and was purified by HPLC (0 to 15% acetonitrile andwater, using 0.1% TFA as modifier). Yield 98.2 mg, 86.6%. Ions found byLCMS: [(M+2H)/2]⁺=581.8, [(M+3H)/3]⁺=388.2.

Example 78. Synthesis of Conjugate 19

This conjugate was prepared analogously to Example 62 (Conjugate 13a) byPEG4-azido-Fc (SEQ ID NO: 35, Example 61) and Int-21 (Example 78). MaldiTOF analysis of the purified final product gave an average mass of56,548. Da (DAR=2.1). Yield 39.7 mg, 39.7%.

Example 79. Synthesis of Int-22

Step a

To a mixture of ether zanamivir acid (1.8 g, 2.8 mmol Example 31) andpropargyl-PEG4-amine (0.82 g, 3.5 mmol. 1.2 eq.) in dichloromethane (50mL), was added EDC (1.0 g, 5 mmol), HOBt (0.65 g, 5 mmol), and DIEA (1.4ml, 10 mmol). The resulting solution was stirred at room temperatureovernight, concentrated, and purified by reverse phase liquidchromatography (RPLC) using an Isco COMBIFLASH® liquid chromatographeluted with 10% to 100% acetonitrile and water with no 0.1% TFA asmodifier. Yield of products 1.35 g, 50.2%. Ion(s) found by LCMS:M+H=830.4.

Step b

Product from the previous step (1.35 g, 1.6 mmol) was treated withtrifluoacetic acid (20 mL) for 30 min at room temperature. The resultingsolution was concentrated, dissolved in water (10 mL) and MeOH (10 mL),then treated with a lithium hydroxide (120 mg, 5 mmol) solution in water(10 mL). The reaction was stirred 10 min then quenched with 0.5 mlacetic acid. The reaction was concentrated and purified by reverse phaseliquid chromatography (RPLC) using an Isco COMBIFLASH® liquidchromatograph eluted with 0% to 50% acetonitrile and water, using 0.1%TFA as the modifier. Yield of the products 510 mg, 52.8%. Ion(s) foundby LCMS: M+H=604.3.

Example 80. Synthesis of Conjugate 20

This conjugate was prepared analogously to Example 62 (Conjugate 13a) byPEG4-azido-Fc (SEQ ID NO: 35, Example 61) and Int-22 (Example 79). MaldiTOF analysis of the purified final product gave an average mass of55,508. Da (DAR=2.3). Yield 37.0 mg, 37.0%.

Example 81. Synthesis of Int-23

Step a

To a solution of N-Boc-1,4-diaminobutane (941.5 mg, 5 mmol) in anhydrousTHF (10 ml) was added sodium carbonate (1.06 g, 10 mmol) and3-(Boc-amino)propylbromide (1.43 g, 6 mmol). The resulting mixture washeated at 50° C. for 1 day. The salt was then filtered, and concentratedby rotary evaporation. The residue was purified by RPLC (100 g, 5 to 50%acetonitrile and water, using 0.1% TFA as modifier). Yield 1.35 g,58.8%. Ion found by LCMS: [M+H]⁺=346.0.

Step b

To a solution of propargyl PEG-4 acid (781 mg, 3 mmol) and HATU (1.14 g,3 mmol) in anhydrous DMF (3 ml) was added DIPEA (390 mg, 3 mmol),followed by the addition of the solution step-a product (863.8 mg, 2.5mmol) and DIPEA (390 mg, 3 mmol) in anhydrous DMF (3 ml). The reactionmixture was stirred for 30 minutes, then directly purified by RPLC (100g, 5 to 80% acetonitrile and water, using 0.1% TFA as modifier). Yield1.19 g, 81%. Ion found by LCMS: [M+H]⁺=588.3.

Step c

The step-b product (1.19 g, 2.02 mmol) was dissolved in anhydrous THF (6ml). 4N HCl solution in dioxane (4.5 ml) was added, and the reactionmixture was stirred for 1 day. It was then concentrated by rotaryevaporation. The residue was extracted with water (3 ml×3) and ethylacetate (15 ml). The combined aqueous layers were lyophilized. Yield 940mg, quantitative yield. Ion found by LCMS: [M+H]⁺=388.3.

Step d

To a mixture of ether zanamivir acid (315 mg, 0.5 mmol, Example 31) andHATU (209.1 mg, 0.55 mmol) in anhydrous DMF (1 ml) was added DIPEA (65mg, 0.5 mmol). After 5 minutes, a solution of the step-c product (170mg, 0.439 mmol) and DIPEA (130 mg, 1 mmol) in anhydrous DMF (1 ml) wasadded in portions over 20 minutes. The reaction was stirred for anadditional 30 minutes, then directly purified by RPLC (50 g, 30 to 90%acetonitrile and water, using 0.1% TFA as modifier). Yield 208 mg,51.6%. Ion found by LCMS: [(M+2H)/2]⁺=806.7.

Step e

The step-d product (208 mg, 0.129 mmol) was dissolved in TFA (1.5 ml),and the solution was heated at 30° C. for 30 minutes. It was thendirectly purified by RPLC (100 g, 0 to 30% acetonitrile and water, using0.1% TFA as modifier). Yield 134 mg, 72%. Ions found by LCMS:[(M+2H)/2]⁺=606.8, [(M+3H)/3]⁺=405.0.

Step f

To a solution of the step-e product (134 mg, 0.093 mmol) in MeOH (2 ml)was added a solution of LiOH monohydrate (63 mg, 1.5 mmol) in water (2ml). The resulting mixture was stirred for 1.5 hours and thenconcentrated by rotary evaporation. The residue was acidified by 4N HClsolution in dioxane (0.5 ml) and was purified by HPLC (0 to 15%acetonitrile and water, using 0.1% TFA as modifier). Yield 78.4 mg, 62%.Ions found by LCMS: [(M+2H)/2]⁺=566.4, [(M+3H)/3]⁺=378.4.

Example 82. Synthesis of Conjugate 21

This conjugate was prepared analogously to Example 62 (Conjugate 13a) byPEG4-azido-Fc (SEQ ID NO: 35, Example 61) and Int-23 (Example 81). MaldiTOF analysis of the purified final product gave an average mass of56,503. Da (DAR=2.2). Yield 34.4 mg, 34%.

Example 83. Activity of Conjugates 13 to 21 Against High Path H7N9Influenza a, and Two Influenza B Isolates in a Cytopathic Effects (CPE)Assay

A total of 9 conjugates (Table 38) were run at concentrations of 100,10, 1, and 0.1 nM and compared to ribavirin in a CPE assay. The CPEassay followed standard methodology, but briefly, utilized 80-100%confluent monolayers of MDCK cells in a 96-well plate. To these, testarticles were added in triplicate and allowed to incubate at 37° C. (+5%CO₂) until CPE effects were visually apparent. Once CPE was noted, celllayers were stained with 0.011% neutral red for approximately 2 hours.Afterwards, a 50:50 mix of Sorensen citrate buffer/ethanol was added andallowed to incubate for 30 min, then the A₅₄₀ was read on aspectrophotometer and EC₅₀/CC₅₀ values calculated by regressionanalysis.

All conjugates had significant activity against the InfluenzaB/Florida/4/2006 isolate, with an EC₅₀ values ranging from 3.05 to 33.5nm (Table 39). On average, conjugates demonstrated a 275-fold potencyadvantage over ribavirin (EC₅₀ of 3,250 nM). Activity of conjugatesagainst Influenza B/Brisbane/60/2008 was very similar to that seenagainst B/Florida with the exception of conjugate 14, which had an EC₅₀of greater than 100 nM

Importantly, all conjugates were highly active against the high pathInfluenza A/Anhui/1/2013 (H7N9) isolate as well. The average EC₅₀ forall conjugates was 21.2 nM against this isolate (ranging from 12 to 28.5nM), compared to 14,000 nM for ribavirin. Lastly, no direct cytotoxiceffects of the conjugates on MDCK monolayers were detected at theconcentrations tested.

TABLE 38 Conjugates and properties Conjugate Int Fc domain DAR LinkerNotes 13 18 SEQ ID NO: 35 2.2 15 atom ether, dimer 14 7 SEQ ID NO: 352.1 15 atom carbamate, dimer 15 12 SEQ ID NO: 35 2.2 15 atom ether,dimer 16 9 SEQ ID NO: 35 2.1 17 atom ether, dimer 17 19 SEQ ID NO: 352.2 17 atom ether, dimer 18 20 SEQ ID NO: 35 2.2 19 atom ether, dimer 1921 SEQ ID NO: 35 2.1 16 atom ether, dimer 20 22 SEQ ID NO: 35 2.3 naether, monomer 21 23 SEQ ID NO: 35 2.2 14 atom ether, dimer

TABLE 39 Activity of conjugates in a CPE assay against Influenzasubtypes EC50 (nM)* B/Brisbane/ B/Florida/ 60/2008 4/2006 A/Anhui/1/2013(H7N9) (Victoria (Yamagata Conjugate (zoonotic aby way ofn) lineage)lineage) Ribavirin 14000   3300 3250 13 24 21 33.5 14 12 >100 18.5 15  21.5 5.95 4.6 16   28.5 14.5 11 17  28** 11 13 18 31 4.35 3.05 19  14.5 6.8 4.55 20   20.5 12.5 14.5 21   17.5 4.05 3.05 *Average of 2values (VIS + NR) **EC50 of 28 by VIS, >100 by NR

Example 84. Syntheses of Conjugate 22 Step a. Synthesis of PEG4-azidoIVIG

Preparation of 0.05M PEG4-azidoNHS ester solution in DMF/PBS: 6.05 mg ofPEG4-azido NHS ester was dissolved in 0.050 mL of DMF at 0° C. anddiluted to 0.305 mL by adding 0.250 mL of PBS 1× buffer at 0° C. Thissolution was used for preparing other PEG4-azido IVIG with variety ofDAR values by adjusting the equivalents of this PEG4-azido NHS ester PBSsolution.

Preparation of PEG4-azido IVIG: 0.05M PEG4-azidoNHS ester PBS buffersolution (0.301 mL, 15.0 μmol, 5.5 equivalents) was added to a solutionof IVIG (Intravenous Immune Globulin, Baxter)) (407 mg in 9.25 mL of pH7.4 PBS, MW148863 Da, 1.968 μmol) and the mixture was shaken gently for12 hours at ambient temperature. The solution was concentrated using acentrifugal concentrator (100,000 MWCO) to a volume of 1.5 mL. The crudemixture was diluted 1:10 in PBS pH 7.4, and concentrated again. Thiswash procedure was repeated for total of three times. The small moleculereagent was removed with this wash procedure. The concentratedIVIG-PEG4-azide was diluted to 9.25 mL with pH 7.4 PBS 1× buffer andready for Click conjugation. The purified material was quantified usinga NANODROP™ UV visible spectrophotomer (using a calculated extinctioncoefficient based on the amino acid sequence of IVIG). Yield isquantitative after purification.

Step b. Synthesis of Conjugates

Prepared the Click reagent solution: 0.0050M CuSO₄ in PBS buffersolution: 10.0 mg CuSO₄ was dissolved in 12.53 mL PBS×1, than took 6.00mL 0.0050M CuSO₄ solution and added 57.7 mg BTTAA (CAS number1334179-85-9) and 297.6 mg Na Ascorbate to give the Click reagentsolution (0.0050M CuSO4, 0.020M BTTAA and 0.25M Sodium Ascorbate).

A solution of azido functionalized IVIG (140 mg, 3.17 mL, 0.936 μmol,IVIG-Linker-1-Azide) was added to a 15 mL centrifuge tube containingalkyne derivatized small molecule (8.4 mg, 0.00618 mmol, 6.6 eq,described in Example 60). After gently shaking to dissolve all solids,1.50 mL of above click reagent solution of (L-ascorbic acid sodium, 0.25M, 74.2 mg, 0.374 mmol, copper (II) sulfate 0.0050M, 1.2 mg, 0.0075mmol, and BTTAA 0.020M, 12.9 mg, 0.0300 mmol). The resulting mixture wasgently shaken overnight. It was purified by affinity chromatography overa Protein A column, followed by size exclusion chromatography (seeconjugate purification protocol in Example 10). Maldi TOF analysis ofthe purified final product gave an average mass of 151873 Da (DAR=2.7).Yield 51.0 mg, 36% yield.

Example 85. Syntheses of Conjugate 23

Conjugate 23 was prepared analogously to Conjugate 22, substituting theappropriate alkyne functionalized small molecule (Int-23 described inExample 81) in the click conjugation step.

Example 86. Syntheses of Conjugate 24

Conjugate 24 was prepared analogously to Conjugate 22, substituting theappropriate alkyne functionalized small molecule (described in Example19) in the click conjugation step.

Example 87. Efficacy of Conjugates 13, 14, and 21 Against Influenza B ina Lethal Mouse Model

Conjugates were evaluated against a lethal Influenza B influenzainfection in female BALB/c mice (Charles River Laboratories, 6-8 weeks).The challenge virus (B/Malaysia/2506/04) is a mouse-adapted isolatecapable of causing lethal infections in mice. The experiment comprised11 groups of 5 mice. At day 0, all mice were challenged with virus at 3×the LD₉₅ by intranasal inoculation in a volume of 30 μl (approx. 1E4 permouse), after being anesthetized with a mixture of ketamine/xylazine(150 and 10 mg/kg respectively).

All groups received a single IV treatment, 2 hours post viral challengeof test article, vehicle (PBS), or Fc only control (hIgG1 Fc). The studyevaluated Int-18, Int-7, and Int-23 conjugated to identical Fc monomers(conjugates 13, 14, and 21, respectively), tested at 3.0, 1.0, and 0.3mg/kg. Mice were monitored for 2 weeks and animals exceeding 20% bodyweight loss, or were found moribund, were scored as a mortality.

All mice treated with vehicle or the Fc only control, reached mortalityby day 7. In contrast, mice receiving conjugates 13, 14, and 21 werefully protected after receiving a single IV dose at 0.3 mg/kg (Table40). As expected, groups receiving the conjugates at 1.0 or 3.0 mg/kgwere fully protected as well. The potency of all conjugates againstInfluenza B was further supported by the daily body measurements (Table41), which show a less than 5% transient drop across the entire studyfor any conjugate treated group. The activity of conjugates 13, 14, and21 is comparable by dose to the activity of conjugate 6 againstInfluenza A H1N1 and H3N2 subtypes. Since conjugates, 6 and 14 haveidentical targeting moieties (corresponding to Int-7), a singleconjugate may be active against the dominant seasonal influenza types(Influenza A (H1N1), Influenza A (H3N2), and Influenza B).

TABLE 40 Mortality data at study end (day 14). Average of 5 mice;p-values calculated by Log-rank (Mantel-Cox) test Significance toCompound Dosage (mg/kg) % Survival vehicle (p-value) Vehicle (PBS) na 0na Fc alone 3.0 0 p = 1 Conjugate 13 0.3 100 (0.0027) 1.0 100 (0.0027)3.0 100 (0.0027) Conjugate 14 0.3 100 p = 0.0027 1.0 100 p = 0.0027 3.0100 p = 0.0027 Conjugate 21 0.3 100 (0.0027) 1.0 100 (0.0027) 3.0 100(0.0027)

TABLE 41 Mouse body weight data (% BW relative to day 0). Average of 5mice; *data not included once the first animal reaches mortality withina group Fc Conjugate 14 Day post Vehicle alone (mg/kg) challenge (PBS)3.0 0.3 1.0 3.0 0 100.0 100.0 100.0 100.0 100.0 1 98.1 98.1 99.3 99.196.5 2 99.6 96.7 98.6 98.6 98.3 3 96.4 94.9 96.8 97.0 98.7 4 89.8 87.898.4 97.5 97.8 5 83.6 80.8 97.4 97.8 98.0 6 79.0 76.1 96.9 98.3 99.77 * * 99.2 100.5 100.6 8 * * 100.3 101.9 101.1 9 * * 102.5 100.4 100.610 * * 100.6 102.0 101.2 11 * * 100.9 100.9 100.9 12 * * 101.8 101.7101.8 13 * * 101.4 101.7 101.4 14 * * 102.2 102.6 102.2

Example 88. Synthesis of PEG4-azido Fc for Conjugate 25, Conjugate 26,Conjugate 27, and Conjugate 28

Preparation of 0.05M PEG4-azidoNHS ester solution in DMF/PBS×1: 27.40 mgof PEG4-azido NHS ester was dissolved in 0.155 mL of DMF at 0° C. anddiluted to by adding 1.200 mL of PBS 1× buffer at 0° C. This solutionwas used for preparing other PEG4-azido Fc with variety of DAR values byadjusting the equivalents of this PEG4-azido NHS ester PBS×1 solution.

Preparation of PEG4-azido Fc (SEQ ID NO: 48): 0.05M PEG4-azidoNHS esterPBS×1 buffer solution (0.0984 mL, 4.92 μmol, 2.5 equivalents) was addedto a solution of h-IgG1 Fc (SEQ ID NO: 48) (234 mg in 13.605 mL of pH7.4 PBS, MW˜57,976 Da, 4.036 μmol) and the mixture was shaken gently for2 hours at ambient temperature. The solution was concentrated using acentrifugal concentrator (30,000 MWCO) to a volume of 2 mL. The crudemixture was diluted 1:7 in PBS pH 7.4, and concentrated again. This washprocedure was repeated for total of three times. The small moleculereagent was removed with this wash procedure. The concentrated Fc (SEQID NO: 48)-PEG4-azide was diluted to 13.60 mL with pH 7.4 PBS 1× bufferand ready for Click conjugation. The purified material was quantifiedusing a Nanodrop™ UV visible spectrophotomer (using a calculatedextinction coefficient based on the amino acid sequence of h-IgG1).

Preparation of PEG4-azido Fc (SEQ ID NO: 50) was analogous to abovePEG4-azido Fc (SEQ ID NO: 48).

Example 89. Synthesis of Conjugate 25

Preparation of the Click reagent solution: 0.0050M CuSO₄ in PBS×1 buffersolution: 10.0 mg CuSO₄ was dissolved in 12.53 mL PBS×1, than took 10.00mL this CuSO₄ solution and added 86.1 mg BTTAA and 495.3 mg Na Ascorbateto give the Click reagent solution (0.0050M CuSO₄, 0.020M BTTAA and0.25M Sodium Ascorbate). This Click reagent solution will be used forConjugate 25 and Conjugate 26.

A solution of azido functionalized Fc (78.0 mg, 4.535 mL, 1.35 μmol, SEQID NO: 48-PEG4-Azide) was added to a 15 mL centrifuge tube containingalkyne derivatized small molecule (13.2 mg, 8.88 μmol, Int-23). Aftergently shaking to dissolve all solids, the mixture was added with 2.153mL of above Click reagent solution of (L-ascorbic acid sodium, 0.25 M,106.6 mg, 0.538 mmol, copper (II) sulfate 0.0050M, 1.72 mg, 0.0107 mmol,and BTTAA 0.020M, 18.5 mg, 0.0431 mmol). The resulting mixture wasgently shaken for 6 hours at ambient temperature. It was purified byaffinity chromatography over a protein A column, followed by sizeexclusion chromatography (see conjugate purification protocol in Example10). Maldi TOF analysis of the purified final product gave an averagemass of 60973 Da (DAR=2.1). Yield 50.3 mg, 64% yield.

Example 90. Synthesis of Conjugate 26

Preparation of Conjugate 26 was analogous to Conjugate 25 by using thesame batch of PEG₄-azido Fc (SEQ ID NO: 48) and an alkyne-derivatizedsmall molecule (Int-7). Maldi TOF analysis of the purified final productgave an average mass of 61068 Da (DAR=2.2). Yield 49.5 mg, 63% yield.

Example 91. Synthesis of Conjugate 27

Preparation of the Click reagent solution: 0.0050M CuSO₄ in PBS×1 buffersolution: 10.0 mg CuSO₄ was dissolved in 12.53 mL PBS×1, than took 12.00mL this CuSO₄ solution and added 103.3 mg BTTAA and 594.3 mg NaAscorbate to give the Click reagent solution (0.0050M CuSO4, 0.020MBTTAA and 0.25M Sodium Ascorbate). This Click reagent solution will beused for Conjugate 28.

A solution of azido functionalized Fc (80.0 mg, 4.535 mL, 1.38 μmol, SEQID NO: 50-PEG4-Azide) was added to a 15 mL centrifuge tube containingalkyne derivatized small molecule (13.5 mg, 9.10 μmol, Int-23). Aftergently shaking to dissolve all solids, the mixture was added with 2.21mL of above Click reagent solution of (L-ascorbic acid sodium, 0.25 M,109.3. mg, 0.552 mmol, copper (II) sulfate 0.0050M, 1.76 mg, 0.0110mmol, and BTTAA 0.020M, 19.0 mg, 0.0441 mmol). The resulting mixture wasgently shaken for 6 hours at ambient temperature. It was purified byaffinity chromatography over a protein A column, followed by sizeexclusion chromatography (see conjugate purification protocol in Example10). Maldi TOF analysis of the purified final product gave an averagemass of 61447 Da (DAR=2.5). Yield 37.1 mg, 46% yield.

Example 92. Synthesis of Conjugate 28

Preparation of Conjugate 28 was analogous to Conjugate 27 by using thesame batch of PEG₄-azido Fc (SEQ ID NO: 50) and an alkyne-derivatizedsmall molecule (Int-7). Maldi TOF analysis of the purified final productgave an average mass of 61388 Da (DAR=2.4). Yield 44.6 mg, 56% yield.

Example 93. Activity of Conjugate 6 and Conjugate 21 Against High PathInfluenza a (H5N1, H7N9) in a Cytopathic Effects (CPE) Assay

An in vitro assay to determine the potency of conjugates of theinvention was conducted against BSL-3 (high path) influenza A, andgenerally followed standard procedures. Briefly, differentconcentrations of conjugates were mixed with virus (approximately 250TC_(ID50)) and allowed to incubate @35° C. for one hour. Afterincubation, the mixture was added to an 80-90% confluent monolayer ofMDCK cells. After a 90 minute incubation cells were washed andconjugates re-applied. The monolayer was subsequently overlayed withcarboxymethylcellulose to minimize viral spreading and allowed toincubate for two days. After two days of culture cells were washed withPBS and fixed with 10% formalin. After fixation the MDCK monolayer waspermeabilized with Triton X-100 and immunostained with a mouse mAbagainst influenza nucleoprotein. Monolayers were read, and the stainedarea per well was calculated to determine EC_(50/100) values.

The results of the study are summarized in Table 42 and demonstrate thepotency of Conjugate 6 and Conjugate 21 against highly pathogenicstrains with pandemic potential. Importantly, both conjugates generatedEC₁₀₀ values at, or below, 15 nM against four H5N1 and one H7N9 isolate.In contrast, oseltamivir had an EC₁₀₀ of approximately 15 nM onlyagainst one isolate (A/Vietnam/1194/2004) and values ranging from 125to >1000 nM against the other high path strains. These results suggestthat the potential of Conjugate 6 and Conjugate 21 to treat pandemicscaused by highly-virulent influenza to be superior to that ofoseltamivir.

TABLE 42 In vitro activity of Conjugate 6 and Conjugate 12 against highpath influenza isolates High Path Influenza EC100 (nM) (CPE in MDCKcells) H5N1 Vehicle Conjugate 6 Conjugate 21 OseltamivirA/Indonesia/5/2005 N/A ~15 <15  ~200-500 (clade 2.1) A/Vietnam/1194/ N/A~15 <15   ~15 2004 (clade 1) A/turkey/Turkey/1/ N/A <15 <15  ~125-2502005 (clade 2.2) A/Hong Kong/156/ N/A ~15 <15 >1000 1997 (clade 0) H7N9A/Anhui/01/2013 N/A <15 <15  ~500-1000 H1N1 (positive control)A/Netherlands/602/ N/A <15 <15 ~1000 2009

Example 94. Activity of Conjugate 6 and Conjugate 21 Against Influenza a(H1N1) at Different Multiplicities of Infection (MOI) in a CytopathicEffects (CPE) Assay

MDCK cells were seeded at 4×10⁴ cells/well in MEM media in 96 well plate(TC-treated) and incubated at 37° C. 5% CO₂ for 18-24 h. Test articles(Zanamivir, Oseltamivir, Baloxavir, Conjugate 6, and Conjugate 21) atdose-range between 1.93-10000 nM were incubated with influenzaA/WSN/1933 at an multiplicity of infection (MOI) of virus:cell between0.001-1 for 1 h at room temperature (RT). After 1 h, pre-incubated virusand test article were added to 90-100% confluent monolayer of MDCK cellsand incubated for 1 h at RT.

After 1 h, MEM media supplemented with L-glutamine andpenicillin/streptomycin was added to wells. Infected cells wereincubated at 37 C, 5% CO₂ for 72 h. CPE was determined after fixing andstaining cells with crystal violet. EC50 was calculated with non-linearregression analysis using Graphpad Prism 6 software. The results of theCPE assay provided in Table 43 indicate that Conjugate 6 and Conjugate21 outperform standard of care agents in vitro, particularly at highMOIs.

TABLE 43 In vitro activity of Conjugate 6 and Conjugate 12 againstinfluenza A (H1N1) at different multiplicities of infection EC₅₀ (nM)H1N1 A/WSN/1933 (CPE in MDCK cells) MOI 0.001 MOI 0.01 MOI 0.1 MOI 1Zanamivir 137 938 >10000 >10000 Oseltamivir 525 1896 >10000 >10000Baloxavir 5 4 79 >100 Conjugate 6 9 15 >100 >100 Conjugate 12 0.6 2 7 10

Example 95. Efficacy of Conjugate 6 Against Influenza a (H1N1) in aLethal Severe Combined Immunodeficiency Mouse Model

Conjugates were evaluated against a lethal Influenza A infection in maleBALB/c Severe Combined Immunodeficiency (SCID) mice (Stock #001803;Jackson Laboratories, 6-8 weeks old). The challenge virus (A/PuertoRico/08/1934) is a mouse-adapted isolate capable of causing lethalinfections in mice. The experiment comprised 5 groups of 5 mice each. Atday 0, all mice were challenged with virus at 3× the LD₉₅ by intranasalinoculation in a volume of 30 μl (approx. 1E3 per mouse), after beinganesthetized with a mixture of ketamine and xylazine (150 and 10 mg/kg,respectively).

All groups received a single IV treatment of conjugate 6, 2 hours postviral challenge of test article, vehicle (PBS), or Fc only control(hIgG1 Fc). The study evaluated 3 different dose concentrations ofconjugate 6 (0.3, 1.0, or 3.0 mg/kg). Mice were monitored for 5 weeksand animals exceeding 20% body weight loss, or who were found moribund,were scored as a mortality. Body weights were also recorded to monitorthe general health of the animals.

All mice treated with vehicle, or the Fc only control, reached mortalityby week 2. In contrast, mice receiving conjugate 6 were fully protectedafter receiving single IV doses of 1 or 3 mg/kg for the duration of thestudy (FIG. 56, Table 44). When dose of conjugate 6 was lowered to 0.3mg/kg survival dropped to 20% by study end. The 0.3 mg/kg dose was fullyprotected for 3 weeks. The potency of conjugate 6 in this model ofsevere immunodeficiency was further supported by body weight data (FIG.57, Table 45). Groups receiving conjugate 6 at the 1 or 3 mg/kg doseconcentrations demonstrated no more than a transient body weight loss ofless than 3% over the entire course of the study. Furthermore, at studyend both dose groups showed a net gain in weight (7.5 and 2.2%,respectively). The group dosed with the lowest concentration ofconjugate 6 (0.3 mg/kg) had less than a 4% transient loss of body weightover the first 3 weeks of the study before showing signs of infection inweek 4, which ultimately resulted in death for four of five animals.

Collectively these data demonstrate the potency of conjugate 6 byprotecting lethally challenged mice with single IV doses of conjugate aslow as 1 mg/kg. Additionally, this protection was long lasting,extending over the 5 week duration of the study. This was accomplishedin an extreme model of immunodeficiency in mice completely lacking T & Bimmune cells, which are essential in clearing influenza infections. Thisdata supports the use of conjugate 6 to treat both immune competent anddeficient patient populations.

TABLE 44 Mortality of study dose groups per week % Survival (Day postviral challenge) Test article 7 14 21 28 35 Vehicle 80 0 0 0 0 Fc only80 0 0 0 0 Conjugate 6 (3 mg/kg) 100 100 100 100 100 Conjugate 6 (1mg/kg) 100 100 100 100 100 Conjugate 6 (0.3 mg/kg) 100 100 100 80 20

TABLE 45 Average group body weight for study animals over 35 days oruntil first death within a group Conjugate 6 dose Day Vehicle Fc only 3mg/kg 1 mg/kg 0.3 mg/kg 0 100 100 100 100 100 1 99.5 99.8 99.6 99.4 98.62 98.8 99.2 99.9 99.1 98.9 3 97.7 96.8 101 100.6 97.9 4 94.4 95.4 99.498.4 98.5 5 93.7 90.7 100.6 97.9 99.3 6 88.7 83.9 101.2 98.7 98.1 7 81.177.6 101.8 98 96.9 8 101.3 99.7 98.9 9 102.6 100.7 98.8 10 101.6 99.899.4 11 101.6 99.7 100.3 12 101.8 100.2 100.7 13 102.3 101 100.7 14103.9 100.5 99.9 15 104.3 101.8 102.3 16 104.3 102 101.3 17 104.3 100.6102 18 105.4 103.9 100.5 21 105.7 105.9 96.8 22 105.5 103.4 93.9 23105.6 106.9 91 24 108.4 102.9 25 106.4 102.7 26 108.4 103.9 27 108.6103.5 28 109.1 104.7 29 108.96 104.5 30 109 105.8 31 108.2 104.9 35107.5 102.2

Example 96. Conjugate 6 Dose-Dependent Viral Clearance in Lungs

Efficacy studies were conducted in 6-8 weeks female BALB/c mice (CharlesRiver) challenged intranasally with 3×10² PFU/mouse (3× the LD₉₅) ofmouse-adapted influenza A/Puerto Rico/8/1934 (H1N1). Conjugate 6 orhuman IgG1 Fc control was administered as a single intravenous (IV) dose2 h post-challenge at 0.1-3 mg/kg. Oseltamivir was dosed orally, twicedaily for 4 days starting 2 h post-infection at 5 or 15 mg/kg. Bodyweights (BW) were recorded for 4 days. At 4 days post-infection, micewere sacrificed by CO₂ and both lung lobes were harvested. Lungs werehomogenized with 1 mm silica beads in 1 mL PBS using a MagNA Lyser(Roche). Homogenization was carried out at 6,000 rpm for 60 s andchilled on ice for 5 min in-between runs. After lung homogenizationtubes were centrifuged for 10 min at 600×g and supernatant wastransferred into new tube.

To determine the viral burden in lungs (measured as Plaque Forming Units(PFUs)), supernatants of lung homogenate were diluted in infectionbuffer ranging from 10-1 to 10-6. 100 μL of virus dilutions were addedto confluent monolayer of MDCK cells in 24 well plates and incubated for1 h at room temperature with rocking every 15 min. After removing thevirus, liquid overlay media containing Avicel was added to MDCK cells.Cells were incubation at 37° C. 5% CO₂ for 40 h. After incubation, themedia was removed and cells were stained with crystal violet toenumerate plaques. PFUs were calculated relative to weight of the lung(PFU/g lung).

The results of this study demonstrate that low doses of conjugate 6rapidly lower the viral burden orders of magnitude better thanOseltamivir (TAMIFLU®) (FIG. 58, Table 46). This observation hasclinical significance since severe influenza infections are caused bythe virus moving from an initial upper respiratory tract infection tothe lungs.

TABLE 46 Viral burden on day 4 post-infection Log reduction Logreduction Test article [mg/kg] (PFU/mL) (PFU/g) PBS [0] 0.00 0.00 hIgG1Fc [3] −0.04 0.26 Oseltamivir [5] 0.24 0.43 Oseltamivir [15] 0.52 0.75Conjugate 6 [0.1] 0.60 0.79 Conjugate 6 [0.3] 1.33 1.55 Conjugate 6 [1]2.25 2.34 Conjugate 6 [3] 3.20 3.40

Example 97. Conjugate 6 Dose-Dependent Reduction in InflammatoryCytokines in Lungs

Efficacy studies were conducted in 6-8 weeks female BALB/c mice (CharlesRiver) challenged intranasally with 3×10² PFU/mouse (3× the LD₉₅) ofmouse-adapted influenza A/Puerto Rico/8/1934 (H1N1). Conjugate 6 orhuman IgG1 Fc control was administered as a single intravenous (IV) dose2 h post-challenge at 0.1-3 mg/kg. Oseltamivir was dosed orally, twicedaily for 4 days starting 2 h post-infection at 5 or 15 mg/kg. Bodyweights (BW) were recorded for 4 days. At 4 days post-infection, micewere sacrificed by CO₂ and both lung lobes were harvested. Lungs werehomogenized with 1 mm silica beads in 1 mL PBS using a MagNA Lyser(Roche). Homogenization was carried out at 6,000 rpm for 60 s andchilled on ice for 5 min in-between runs. After lung homogenizationtubes were centrifuged for 10 min at 600×g and supernatant wastransferred into new tube.

For cytokine analysis, supernatants of lung homogenate were seriallydiluted 2-fold in 96 well plate. Cytokine levels for INF-γ, TNF-α, IL-6,MIP-1α and MCP-1 were determined by ELISA according to manufacturer'sinstructions (R&D Systems).

Morbidity and mortality from severe influenza is ultimately caused byvirally induced influx of pro-inflammatory cytokines in the lungs. Onepotential concern of using an Fc-conjugate to treat influenza is whetherthe Fc fragment would exacerbate cytokine induced inflammation. Theresults of the H1N1 lethal infection model show just the opposite: aconjugate 6 dose-dependent decrease in pro-inflammatory cytokines (e.g.,TNFα and IL-6) in infected lung tissues (FIG. 59, Table 47).

TABLE 47 Cytokine response on day 4 post-infection Fold-shift comparedto unifected control Test article [mg/kg] INFy TNFa IL-6 MCP-1 MIP-1aPBS [0] 0.4 2.2 4.6 16.3 16.4 hIgG1 Fc [3] 0.5 2.2 6.2 20.2 16.8Oseltamivir [5] 0.2 1.7 4.0 11.7 7.5 Oseltamivir [15] 0.2 1.8 3.5 10.56.1 Conjugate 6 [0.1] 0.3 1.5 3.0 10.8 7.9 Conjugate 6 [0.3] 0.2 1.2 1.96.7 7.4 Conjugate 6 [1] 0.2 1.1 1.9 6.1 3.6 Conjugate 6 [3] 0.8 1.1 1.42.8 2.6 Uninfected 1.0 1.0 1.0 1.0 1.0

Example 98. In Vivo Conjugate 6 Plasma Sample Analysis. Comparison of PKin CD-1 and BALB/Severe Combined Immune Deficient Mice

Conjugate 6 in plasma samples were quantified by a neuraminidase capturedetection ELISA. Briefly, molecules were captured on neuraminidasecoated plates and then detected using a HRP-conjugated anti-human IgG-Fcantibody. Protein concentration was calculated in GraphPad Prism using4PL non-linear regression of Conjugate 6 standard curves. A moredetailed method description is provided below.

Nunc Maxisorp 96-well plates (Cat No. 12-565-136, ThermoFisher) werecoated with 0.1 U/well neuraminidase from A/California/04/2009 (H1N1)(11058-VNAHC, Sino Biological) in 1×KPL coating buffer (5150-0041,SeraCare). Plates were incubated at room temperature for 1 hr on anorbital plate shaker (500 rpm). Serial dilutions of the plasma sampleswere plated and incubated at room temperature for 2 hours (samplediluent: 0.5% BSA in PBS 0.025% Tween 20+naïve mouse plasma finalconcentration of 1:2,500). Conjugate 6 standard curves ranging from0.230 to 500 ng/mL, in duplicate were run on each plate. Following the 2hr incubation, plates were washed 5× in 300 μL PBS with 0.05% Tween 20.Conjugate bound to neuraminidase on the plates was then probed with anHRP conjugated anti-human IgG Fc F(ab′)2 (709-036-098, Jackson) diluted1:1,000 in sample diluent for 1 hr at room temp. Plates were then washed8× in 300 uL PBS with 0.05% Tween 20 and developed with TMB substratefor 7-8 minutes. The reaction was stopped with 1N H₂SO₄. Absorbance wasread at 450 nm. Conjugate 6 in plasma samples was interpolated usingGraphPad Prism Version 6 following nonlinear regression analysis(Sigmoidal, 4PL analysis) of the standard curves.

PK Profiles, CD-1 vs BALB/c SCID Mouse

Conjugate 6 administered intravenously to SCID and CD-1 (immunecompetent) mice at 5 mg/kg demonstrated similar PK profiles (FIG. 60).Concentrations were comparable at the sampled time points. The two-phasePK profiles comprise 24-hour distribution phases followed by a shallowelimination phase. Conjugate 6 plasma levels remained high (˜10 μg/ml)relative to C_(max) levels over the one-week course of the study.

Example 99. Synthesis of the Propargyl Diamine Central Linker

Step a

A solution of 2-(2-Boc-Aminoethoxy)ethanol (16.0 g, 78.0 mmol) and CBr₄(31.0 g, 93.5 mmol) in DCM (100 mL) at 0° C. was treated with PPhb (24.5g, 93.5 mmol) slowly over 15 minutes (exothermic). During the course ofthe addition the internal temperature was kept below 30° C. Afteraddition of PPh₃ the reaction was stirred overnight at room temperature.The crude reaction was concentrated to an oil then purified by normalphase chromatography, eluting with 10% ethyl acetate/hexanes to 80%ethyl acetate/hexanes. Fractions containing oil droplets on the insideof the collection tubes were combined and concentrated to a colorlessoil. Yield 18.1 g, 86%.

Step b

A solution of the step-a product (10 g, 37.3 mmol), benzylamine (1.60 g,14.9 mmol), and K₂CO₃ (6.19 g, 44.8 mmol) in DMF (20 mL) were heated inan oil bath at 75° C. for 8 h. The mixture was filtered, concentratedand purified by RPLC (5% ACN/water to 100% ACN). Yield 6.8 g, 95%.

Step c

To a solution of the step-b product (5.35 g, 8.98 mmol) in CHCl₃/EtOH(1:20, 100 mL) was added 20% Pd(OH)₂/C (1.26 g, 1/80 mmol). The reactionwas stirred overnight under hydrogen balloon at ambient temperature. Thereaction mixture was filtered through a Celite pad. The solvents wereremoved and carried to the subsequent step without purification.

Step d

The step-c product was re-dissolved in 20 mL of DMF/dichloromethane(1:5). To this free amine solution propargyl PEG₄ acid (2.36 g, 8.98mmol), EDCI (2.57 g, 13.5 mmol), HOAt (1.83 g, 13.5 mmol) and Hunig'sbase (3.13 mL, 18.0 mmol) were added. The reaction mixture was stirredfor four hours, then concentrated and purified by RPLC (10% ACN/water to60% ACN/water. Yield 4.00 g, 70% over two steps. Ions found by LCMS:[M-Boc+H]⁺=534.2, [M+H]⁺=634.2.

Step e

The step-d product (4.00 g, 6.31 mmol) was treated with 4N HCl indioxane (30 mL) for 2 hours. Extra HCl and dioxane were removed byrotary evaporation, and the remaining was further dried under highvacuum to give Int-10 as 2HCl salt. Yield 3.15 g, 99%. Ion found byLCMS: [M+H]⁺=434.2.

Example 100. Synthesis of Int-7a (C7-C7 Isomer)

Step a

Methyl5-acetamido-7,8,9-O-triacetyl-2,6-anhydro-4-azido-3,4,5-trideoxy-D-glycero-D-galacto-non-2-enonate(30 g, 65.7 mmol) was dissolved into methanol (100 mL) and combined withLindlar catalyst (15 g). The resulting mixture was flushed with hydrogenand stirred for 5 hours, flushing hydrogen through the headspace withhydrogen every 30 minutes. After complete reaction as determined byHPLC, the catalyst is filtered through celite. The filtrate was used inthe next step.

Crude amine from the previous step (18.9 g, 43.8 mmol) was treated withN,N′-bis-boc-1-guanylpyrazole (14.3 g, 46.0 mmol), and DIEA (9.9 ml,57.0 mmol) in methanol (100 mL). The resulting solution was stirred atroom temperature until all starting material was consumed as determinedby LCMS (˜30 min). The solution was concentrated to a foam and storedunder high vacuum overnight then used without further purification inthe next step.

Crude tri-acetate from the previous step (43.8 mmol) was dissolved in100 ml dry methanol, then treated with sodium methoxide in methanol (1.9mL, 25% solution in methanol, 8.76 mmol) at room temperature. Progressof reaction was monitored by LCMS which was complete after 10 minutes.The reaction was quenched with 1N HCl to a pH of 7. The resultingsolution was concentrated and purified by reverse phase liquidchromatography (RPLC) using an Isco COMBIFLASH® liquid chromatographeluted with 10% to 100% acetonitrile and water. No TFA modifier was usedfor this purification. Yield of product 15.6 g, 65%.

Step b

A mixture of the step-a product (5.47 g, 10 mmol) and DMAP (1.222 g, 10mmol) was dissolved in anhydrous THF (30 ml). After cooling in anice-water bath, the solution was slowly treated with1,1′-carbonyldiimidazole (2.6 g, 16 mmol), then stirred for 30 minutesat 0° C., followed by heating at 60° C. for 2 hours. It was then cooledto room temperature and extracted with water (50 ml) and EtOAc/hexane(1:1, 100 ml). The organic layer was washed with water (50 ml×3), driedover Na₂SO₄ and concentrated by rotary evaporation. The white foamproduct was further dried under high vacuum and carried to thesubsequent step without further purification. Ion found by LCMS:[M+H]⁺=573.2.

Step c

A reaction flask containing the step-b product was vacuum flushed withnitrogen and dissolved in anhydrous DCM (50 ml), then cooled in anice-water bath. To the cooled solution was added DMAP (4.89 g, 40 mmol),followed by 4-nitrophenylchloroformate (6.05 g, 30 mmol) was added inportions over 20 minutes. The solution was stirred at 0° C. then warmedto room temperature for 1 hour. LCMS shows starting material 1 hr soadditional DMAP (1.22 g, 10 mmol) and 4-nitrophenylchloroformate (1 g, 5mmol) were added. The reaction was continued for 4 hours, then purifiedwith two silica gel columns (220 g, pre-wet by 20% EtOAc and hexane) andeluted with 20% to 80% EtOAc and hexane. Yield 4.42 g, 59.9% for twosteps. Ion found by LCMS: [M+H]⁺=738.2

Step d

To a solution of the step-c product (3.2 g. 4.34 mmol) in anhydrous DCM(3 ml) was added in-portions over 30 minutes a mixture of propargyldiamine central linker (1.26 g, 2.5 mmol, described in Example 99) andDIPEA (1.68 g, 13 mmol) in anhydrous DMF (5 ml). The reaction wasstirred at room temperature for 2 hours. It was then concentrated andpurified by RPLC (30% to 90% acetonitrile and water, no TFA modifier).Ions found by LCMS: [(M+2H)/2]⁺=815.8, [(M-Boc+2H)/2]⁺=765.8,[(M-2Boc+2H)/2]⁺=716. Yield 3.33 g, 94.2%.

Step e

The step-d product (3.33 g, 2.04 mmol) was dissolved in DCM (5 ml) andTFA (5 ml) then stirred at 35° C. for ˜6 hours. The reaction wasmonitored by LCMS. When complete the solution was concentrated andpurified by RPLC (5 to 30% acetonitrile and water, no TFA). Ions foundby LCMS: [(M+2H)/2]⁺=615.8, [(M+3H)/3]⁺=411. Yield 2.29 g, 91.3%.

Step f

The step-e product (61.5 mg, 0.05 mmol) was dissolved in MeOH/water(1:1, 0.6 ml). After the solution was cooled to −6° C. (salt/ice bath),1.0 M LiOH (0.3 ml, 0.3 mmol) was added drop-wise and the reaction wasstirred for 30 minutes. It was then quenched to pH˜7.0 with 4N HCl indioxane solution (75 μl) and directly purified by prep HPLC (Isco ACCQprep, Luna 5 μm C18(2) 100 Å LC column 100 mm×30 mm; Gradient: 0%acetonitrile/water for 2 min, then 0% to 15% acetonitrile/water over 12min, then isocratic at 15% acetonitrile for 10 min, using 0.1% TFA).Yield 45 mg, 65.3%. Ions found by LCMS: [(M+2H)/2]⁺=575.8,[(M+3H)/3]⁺=384.2. Analytical retention time: 6.013 min. Conditions:Phenomemex Gemini HPLC column, 3 μm WX-C18 110 Å, 100 mm×3 mm, elutedover 25 minutes with 5-95% acetonitrile and water gradient, using 0.1%TFA.

Example 101. Synthesis of Int-7b (C7-C9 Isomer)

The C7-C9 heterodimer (Int-7b) was prepared analogously to Int-7a (C7-C7isomer, Example 100), with the exception that the reaction is conductedat 0° C. and is monitored by HPLC (retention time: 6.112 min.Conditions: see Example 100, synthesis of Int-7a and stopped when theC7-C9 isomer predominates (˜3 h). This isomer is isolated using the sameconditions that were used to isolate Int-7a. Ions found by LCMS:[(M+2H)/2]⁺=575.8, [(M+3H)/3]⁺=384.2.

Example 102. Synthesis of Int-7c (C9-C9 Isomer)

Int-7c (C9-C9 isomer) was prepared analogously to Int-7a (C7-C7 isomer,Example 100), with the exception that the reaction conducted at 0° C.and is monitored by HPLC (retention time: 6.232 min. Conditions: seeExample 100, synthesis of Int-7a and stopped when the C9-C9 isomerpredominates (˜6 h). This isomer is isolated using the same conditionsthat were used to isolate Int-7a. Ions found by LCMS: [(M+2H)/2]⁺=575.8,[(M+3H)/3]⁺=384.2.

Example 103. Synthesis of Int-7 (Acetonide Route)

Step a

Methyl5-acetamido-7,8,9-O-triacetyl-2,6-anhydro-4-azido-3,4,5-trideoxy-D-glycero-D-galacto-non-2-enonate(10.0 g, 22 mmol) was dissolved in 60 ml dry methanol, then treated with20 ml sodium methoxide in methanol (0.5 M in methanol, 10 mmol) whilecooling in an ice-water bath. Progress of the reaction was monitored byLCMS, which was complete after 2 hours. The pH of the reaction solutionwas then adjusted to a value of 5 to 6 by using Amberlite IRN-77 ionexchange resin. The mixture was filtered to remove the resin andevaporated to dryness under vacuum. The resulting oil was used for nextstep without further purification, Ion(s) found by LCMS: M+H=331.1.

Step b

To a solution of the products from the previous step in 70 ml of acetonewere added 30 ml of 2,2-dimethoxypropane and p-toluenesulfonic acid 1hydrate (400 mg, 2.0 mmol), the resulting solution was stirred at roomtemperature overnight. At the end of this time, sodium bicarbonate (170mg, 2.0 mmol) was added, and the mixture was concentrated to dryness.The resulting residue was used in next step without purification, Ion(s)found by LCMS: M+H=371.2.

Step c

To a solution of material from the previous step in 60 ml of methanolwas added 5.0 g of a Lindlar catalyst. The resulting mixture was flushedwith hydrogen every 30 minutes and stirred for 5 hours. After completereaction as determined by HPLC, the catalyst was filtered off throughcelite. The filtrate was concentrated and used in the next step withoutpurification.

Crude product from the previous step in 60 ml THF was treated withN,N′-bis-boc-1-guanylpyrazole (9.3 g, 30.0 mmol), and DIEA (9.9 ml, 57.0mmol). The resulting solution was stirred at room temperature until allstarting material was consumed as determined by LCMS (4 h). The solutionwas concentrated and purified by flash chromatography eluted with 20% to80% ethyl acetate/dichloromethane. Yield 8.8 g, 59.0% for four steps,Ion(s) found by LCMS: M+H 587.3.

Step d

A reaction flask containing product from the previous step (6.5 g, 11mmol) was vacuum flushed with nitrogen and dissolved in anhydrousdichloromethane (100 ml). After the solution was cooled in an ice-waterbath, DMAP (4.89 g, 40 mmol) was added and stirred to dissolve, then4-nitrophenylchloroformate (5.58 g, 28 mmol) was added in portions,while stirring at 0° C. to room temperature for 1 hour. LCMS showsstarting material 1 hr so additional DMAP (1.22 g, 10 mmol) and4-nitrophenylchloroformate (1.0 g, 5 mmol) were added. The reaction wascontinued for 4 more hours, then concentrated and purified by flashchromatography eluting with 20% to 80% ethyl acetate/dichloromethane.Yield 5.1 g, 59.9%. Ion found by LCMS: [M+H]⁺=752.2

Step e

To a solution of the nitrophenyl carbonate from the previous step (1.8g. 2.3 mmol) in anhydrous dichloromethane (20 ml) was added a mixture ofcentral linker (0.51 g, 1.0 mmol, added in portions over 30 minutes) andDIPEA (1.4 ml, 10 mmol) in anhydrous DMF (20 ml). The reaction wasstirred at room temperature overnight, then concentrated and purified byflash chromatography eluting with 0% to 10% methanol/dichloromethane.Yield 1.35 g, 80%. Ions found by LCMS: [(M+2H)/2]⁺=830.4,[(M-Boc+2H)/2]⁺=780.4, [(M-2Boc+2H)/2]⁺=730.4.

Step f

Product from the previous step (200 mg, 0.2 mmol) was dissolved into 2ml MeOH and 2 ml THF, then treated with a solution of lithium hydroxide(24 mg, 1 mmol) dissolved in 2 ml water. The reaction was stirred for 10min at room temperature at which time HPLC showed the reaction wascomplete. The pH of the reaction solution was adjusted to the value of 5to 6 by using Amberlite IRN-77 ion exchange resin the filtered to removethe resin. The crude product was evaporated to dryness under a vacuumand used in the next step with purification, Ion(s) found by LCMS:[(M+2H)/2]⁺=815.4, [(M-Boc+2H)/2]⁺=765.4, [(M-2Boc+2H)/2]⁺=715.4.

Step g

The product from step g (400 mg, 0.25 mmol) was dissolved into 5 mldichloromethane and 5 ml TFA, and the resulting reaction solution wasstirred at room temperature. The progress of the reaction was monitoredby LCMS. After the completion of the reaction (6 h), the solution wasstripped to dryness and then dissolved in 4 ml water and 4 mlacetonitrile. The resulting solution was stirred for another 2 hour atroom temperature at which LCMS show complete deprotection of theacetonide protecting groups. This mixture was concentrated and purifiedby reverse phase liquid chromatography (RPLC) using an Isco COMBIFLASH®liquid chromatograph eluted with 5% to 40% acetonitrile/water with 0.1%TFA as the modifier. Yield 270 mg, 68.0%. Ion(s) found by LCMS:[(M+2H)/2]⁺=575.8, [(M+3H)/3]⁺=384.2.

Example 104. NMR Results for Int-7a, Int-7b, and Int-7c

NMR of Int-7a

¹H NMR (500 MHz, Methanol-d₄) δ 5.91-5.89 (m, 2H), 5.00-4.96 (m, 2H),4.58-4.53 (m, 2H), 4.42-4.37 (m, 2H), 4.20-4.17 (m, 6H), 4.02-3.97 (m,2H), 3.78-3.49 (m, 28H), 3.29-3.18 (m, 4H), 2.86 (t, J=2.6 Hz, 1H),2.85-2.72 (m, 2H), 1.96 (s, 3H), 1.95 (s, 3H).

¹³C NMR (125 MHz, MeOD) δ 171.73, 170.80, 170.29, 161.95, 156.06,155.08, 154.99, 144.07, 143.93, 116.41, 114.10, 106.03, 105.86, 77.71,74.46, 73.22, 68.62, 68.54, 68.50, 68.38, 68.12, 68.00, 67.94, 67.67,67.64, 67.53, 67.20, 66.96, 65.44, 61.53, 56.17, 49.74, 49.64, 47.37,45.17, 39.16, 39.06, 31.73, 19.96, 19.92.

NMR of Int-7b

¹H NMR (500 MHz, Methanol-d₄) δ 5.91-5.87 (m, 2H), 5.04-4.95 (m, 1H),4.58-4.48 (m, 2H), 4.45-4.37 (m, 3H), 4.20-4.10 (m, 5H), 4.08-3.98 (m,2H), 3.79-3.44 (m, 29H), 3.29-3.18 (m, 4H), 2.88-2.70 (m, 3H), 2.02 (s,3H), 1.96-1.94 (m, 3H).

¹H NMR (500 MHz, DMSO-d₆) δ: 8.28 (d, J=8.4 Hz, 2H, —NH), 7.98 (d,J=11.5 Hz, 2H, —NH), 7.78 (d, J 20=8.7 Hz, 2H, —NH), 7.60 (t, J=8.5 Hz,2H, —NH), 7.20 (bs, 2H, —NH), 7.06 (bs, 2H, —NH), 5.69 (bs, 1H), 5.67(d, J=2.4 Hz, 1H), 4.83 (dd, J=9.2, 2.2 Hz, 1H), 4.45 (dt, J=9, 2.5 Hz,1H), 4.37 (d, J=2.2 Hz, 1H), 4.33-4.24 (m, 2H), 4.13 (d, J=2.5 Hz, 2H),4.05-4.32 (m, 41H), 3.23 (m, 1H), 3.15-3.09 (m, 2H), 3.06-3.02 (m, 2H),2.59 (t, J=6.7 Hz, 2H), 1.91 (s, 3H), 1.78 (s, 3H).

¹³C NMR (125 MHz, MeOD) δ: 173.19, 172.98, 172.25, 171.75, 164.26,163.82, 157.88, 157.55, 156.51, 146.09, 107.21, 106.93, 106.59, 79.22,76.19, 75.92, 74.72, 74.68, 70.12, 70.08, 70.03, 69.99, 69.92, 69.88,69.66, 69.48, 69.12, 69.01, 68.94, 68.73, 68.69, 68.50, 68.19, 67.08,66.93, 66.79, 63.04, 57.68, 51.24, 51.15, 50.13, 48.87, 48.72, 46.72,46.58, 46.23, 40.64, 40.41, 33.21, 21.49, 21.45, 21.40.

NMR of Int-7c

¹H NMR (500 MHz, Methanol-d₄) δ: 5.88 (d, J=2.6 Hz, 2H), 4.50 (dt,J=8.5, 2.7 Hz, 2H), 4.43 (ddd, J=9.7, 3.9, 1.5 Hz, 2H), 4.39 (dd,J=11.5, 2.4 Hz, 2H), 4.25-4.16 (m, 2H), 4.19 (d, J=2.4 Hz, 2H), 4.13(dt, J=11.5, 5.9 Hz, 2H), 4.05 (m, 2H), 3.77 (t, J=6.2 Hz, 2H),3.72-3.55 (m, 22H), 3.51 (m, 4H), 3.35-3.23 (m, 4H), 2.86 (t, J=2.4 Hz,1H), 2.74 (t, J=6.2 Hz, 2H), 2.02 (s, 6H).

¹³C NMR (125 MHz, MeOD) δ: 173.03, 163.76, 157.89, 157.54, 145.60,107.21, 79.28, 76.29, 74.70, 70.11, 70.07, 70.03, 69.92, 69.70, 69.47,68.96, 68.89, 68.72, 68.54, 68.19, 67.05, 66.75, 57.69, 50.09, 48.69,48.08, 46.10, 40.44, 40.38, 33.23, 21.42.

Example 105. Synthesis of Int-60

Step a

To a 0° C. stirring solution of previously prepared Ether-zanamivir acid(1.00 g, 1.586 mmol, Example 31), 2-azidoethylamine hydrochloride (213mg, 1.744 mmol) and DIPEA (1.105 mL, 6.343 mmol) in DMF (8.0 mL), it wasadded HATU (615 mg, 1.618 mmol). The temperature was raised to ambientand stirring was continued until completion. All the volatiles wereremoved per vacuum techniques. The residue was taken up in ethylacetate, washed with a 1 M aqueous solution of sulfuric acid (1×50 mL),then a saturated aqueous solution of sodium bicarbonate (3×20 mL), andbrine (1×50 mL). The resulting organic layers was dried with magnesiumsulfate, filtered, and all the volatiles were removed per vacuumtechniques. In this way, 816 mg of the desired intermediate azide wasobtained in high purity and used in the next step without any furtherpurification. (Ion found by LCMS: [M+H]+=699.2). To a stirring solutionof the described crude material (816 mg, 1.168 mmol), dipropargylamine(54 mg, 0.584 mmol), tris((1-benzyl-4-triazolyl)methyl)amine (31 mg,0.058 mmol), and sodium ascorbate (58 mg, 0.292 mmol) in ethanol (10 mL)and water (5 mL), it was added cupric sulfate (10 mg, 0.061 mmol). Uponcompletion, copper scavenger SiliaMetS TAAcONa (300 mg, loading 0.45mmol/g) was added and stirring was continued for 1 h. The mixture wasfiltered with the aid of dichloromethane. The filtrate was washed with asaturated solution of sodium bicarbonate. The aqueous layer wasadditionally washed with dichloromethane (3 times). The combinedorganics were dried with magnesium sulfate, filtered and concentrated.The residue was purified by silica column using an Isco COMBIFLASH®liquid chromatography eluted with 0% to 100% hexanes and ethyl acetate,followed by 0% to 30% dichloromethane and. Yield 817 mg, 94% yield. Ionsfound by LCMS: [(M+2H)/2]⁺=745.8, [(M+3H)/3]⁺=497.5.

Step b

To a 0° C. stirring solution of step a product (817 mg, 0.548 mmol),propargyl-PEG4-acid (185 mg, 0.712 mmol) and DIPEA (286 uL, 1.644 mmol)in DMF (7.0 mL), it was added HATU (212 mg, 0.544 mmol). The temperaturewas raised to ambient and stirring was continued until completion. Allthe volatiles were removed per vacuum techniques. The residue waspurified by HPLC (0 to 90% methanol and water). Yield 520 mg, 56%. Ionsfound by LCMS: [(M+2H)/2]⁺=866.8, [(M+3H)/3]⁺=578.4.

Step c

A stirring solution of step b compound (520 mg, 0.300 mmol) in2-methyl-2-butene (0.25 mL), dichloromethane (4.0 mL) and TFA (2.0 mL)was stirred until gas evolution ceased. All the volatiles were removedper vacuum techniques. The residue was purified by HPLC (0 to 30%methanol and water, using 0.1% TFA as modifier). Yield 221 mg, 47%. Ionsfound by LCMS: [(M+2H)/2]⁺=666.8, [(M+3H)/3]⁺=444.8.

Step d

To a 0° C. stirring solution of step c product (221 mg, 0.142 mmol) inwater (3.0 mL) it was added lithium hydroxide (20 mg, 0.850 mmol). Thereaction was quenched with acetic acid (120 uL), and all the volitileswere removed per vacuum techniques. The residue was purified by HPLC (0to 20% methanol and water, using 0.1% TFA as modifier). Yield 130 mg,62%. Ions found by LCMS: [(M+2H)/2]⁺=626.8, [(M+3H)/3]⁺=418.2.

Example 106. Synthesis of Conjugate 29

A solution of azido functionalized aglycosylated Fc (SEQ ID NO: 35) inpH 7.4 PBS×1 buffer solution (100 mg, 10 mL, 1.874 μmol) is added to acentrifuge tube containing a pH 7.4 PBS×1 buffer solution (10.50 mL) ofalkyne derivatized small molecule (17 mg, 0.0112 mmol; Example 105,Int-60), cupric sulfate (4 mg, 0.0225 mmol),tris(3-hydroxypropyltriazolylmethyl)-amine (39 mg, 0.0900 mmol), andsodium ascorbate (7.4 mg, 0.375 mmol). The resulting mixture was gentlyshaken overnight. It was purified by affinity chromatography over aprotein A column, followed by size exclusion chromatography (see Example10). Maldi TOF analysis of the purified final product gave an averagemass of 56938 Da (DAR=2.3). Yield 49.9 mg, 49% yield.

Example 107. Synthesis of Int-65

Step a

To a 0° C. stirring solution of previously prepared ether-zanamivir acid(2.00 g, 3.172 mmol, Example 31) and 4-methylmorpholine (0.628 mL, 5.709mmol) in tetrahydrofuran (30 mL) it was added isobutyl chloroformate(0.617 mL, 4.7574 mmol). After 10 minutes, the temperature was increasedto ambient and stirring continued for 20 minutes. The temperature wasdecreased back to 0° C., and sodium borohydride (360 mg, 9.515 mmol) wasadded in one portion, followed by dropwise addition of (10 mL) over 5minutes. Upon completion, the reaction was quenched with acetic acid(2.860 mL, 50 mmol), and after 5 minutes the temperature was raised toambient, while stirring was continued until gas evolution ceased. Allthe volatiles were evaporated and the residue was suspended indichloromethane and filtered. The filtrate was concentrated and theresidue was purified by silica column using an Isco COMBIFLASH® liquidchromatography eluted with 20% to 100% hexanes and ethyl acetate, using3% methanol as a modifier. Yield 1.302 g, 66% yield. Ions found by LCMS:[(M+H)]+=617.2.

Step b

To a 0° C. stirring solution of step a product (1.25 g, 2.027 mmol) andDIPEA (1.095 mL, 6.284 mmol) in dichloromethane (15 mL), it was addedmethanesulfonyl chloride (0.314 mmol, 4.054 mmol). Upon completion, thereaction was treated with water (15 mL). The layers were separated, andthe dichloromethane layer was dried with brine, then magnesium sulfate,and filtered. The solution was concentrated and the residue wasdissolved in DMF (10 mL), and sodium azide (264 mg, 4.054 mmol) wasadded, while temperature was raised to 50° C. Completion was observedafter 18 h, and all the volatiles were evaporated per vacuum techniques.The residue was purified by silica column using an Isco COMBIFLASH®liquid chromatography eluted with 20% to 100% hexanes and ethyl acetate,using 3% methanol as a modifier. Yield 767 mg, 59% yield. Ions found byLCMS: [(M+H)]+=642.2.

Step c

To a stirring solution of step b product (496 mg, 0.773 mmol),dipropargylamine (36 mg, 0.386 mmol),tris((1-benzyl-4-triazolyl)methyl)amine (41 mg, 0.077 mmol), and sodiumascorbate (115 mg, 0.580 mmol) in ethanol (16 mL) and water (8 mL), itwas added cupric sulfate (13 mg, 0.081 mmol). Upon completion, copperscavenger SiliaMetS TAAcONa (600 mg, loading 0.45 mmol/g) was added andstirring was continued for 1 h. The mixture was filtered with the aid ofdichloromethane. The filtrate was washed with a saturated solution ofsodium bicarbonate. The aqueous layer was additionally washed withdichloromethane (3 times). The combined organics were dried withmagnesium sulfate, filtered and all the volatiles were evaporated pervacuum techniques. To a 0° C. stirring solution of the residue,propargyl-PEG4-acid (151 mg, 0.580 mmol)) and DIPEA (337 uL, 1.933 mmol)in DMF (10.0 mL), it was added HATU (220 mg, 0.580 mmol). Thetemperature was raised to ambient and stirring was continued untilcompletion. All the volatiles were removed per vacuum techniques. Theresidue was purified by HPLC (0 to 90% methanol and water). Yield 457mg, 73%. Ions found by LCMS: [(M+2H)/2]+=809.8, [(M+2H-Boc)/2]+=759.8.

Step d

A stirring solution of step c compound (451 mg, 0.279 mmol) in2-methyl-2-butene (0.25 mL), dichloromethane (4.0 mL) and TFA (2.0 mL)was stirred until gas evolution ceased. All the volatiles were removedper vacuum techniques. Ions found by LCMS: [(M+2H)/2]+=609.8,[(M+3H)/3]+=407.0. To a 0° C. stirring solution of the residue intetrahydrofuran (6 mL) and water (6 mL), it was added lithium hydroxide(240 mg, 10.03 mmol). Upon completion, the reaction was quenched withacetic acid (0.638 mL, 11.14 mmol), and all the volatiles were removedper vacuum techniques. The residue was purified by HPLC (0 to 90%methanol and water, using 0.1% TFA as modifier). Yield 209 mg, 67%. Ionsfound by LCMS: [(M+2H)/2]+=569.8, [(M+2H-Boc)/2]+=380.3.

Example 108. Synthesis of Conjugate 30

A solution of azido functionalized aglycosylated Fc (Example 7, SEQ IDNO: 35) in pH 7.4 PBS×1 buffer solution (50 mg, 5 mL, 1.874 μmol) isadded to a centrifuge tube containing a pH 7.4 PBS×1 buffer solution(9.50 mL) of alkyne derivatized small molecule (8.7 mg, 0.0064 mmol,Int-65), cupric sulfate (2 mg, 0.013 mmol),tris(3-hydroxypropyltriazolylmethyl)-amine (22 mg, 0.0508 mmol), andsodium ascorbate (25 mg, 0.127 mmol). The resulting mixture was gentlyshaken overnight. It was purified by affinity chromatography over aprotein A column, followed by size exclusion chromatography (see Example10). Maldi TOF analysis of the purified final product gave an averagemass of 61548 Da (DAR=2.4). Yield 32.33 mg, 67% yield.

Example 109: Synthesis of p-Nitrophenyl Carbonate Zanamivir Intermediate

Step a

Triacetoxy-azido Zanamivir intermediate (10.0 g, 22 mmol) was dissolvedin 60 ml dry methanol, then treated with 20 ml sodium methoxide inmethanol (0.5 M in methanol, 10 mmol) while cooling in an ice-waterbath. Progress of the reaction was monitored by LCMS, which was completeafter 2 hours. The pH of the reaction solution was then adjusted to avalue of 5 to 6 by using Amberlite IRN-77 ion exchange resin. Themixture was filtered to remove the resin and evaporated to dryness undervacuum. The resulting oil was used for next step without furtherpurification, Ion(s) found by LCMS: M+H=331.1.

Step b

To a solution of the products from the previous step in 70 ml of acetonewere added 30 ml of 2,2-dimethoxypropane and p-toluenesulfonic acid 1hydrate (400 mg, 2.0 mmol), the resulting solution was stirred at roomtemperature overnight. At the end of this time, sodium bicarbonate (170mg, 2.0 mmol) was added, and the mixture was concentrated to dryness.The resulting residue was used in next step without purification, Ion(s)found by LCMS: M+H=371.2.

Steps c & d

To a solution of material from the previous step in 60 ml of methanolwas added 5.0 g of a Lindlar catalyst. The resulting mixture was flushedwith hydrogen every 30 minutes and stirred for 5 hours. After completereaction as determined by HPLC, the catalyst was filtered off throughcelite. The filtrate was concentrated and used in the next step withoutpurification.

Crude product from the previous step in 60 ml THF was treated withN,N′-bis-boc-1-guanylpyrazole (9.3 g, 30.0 mmol), and DIEA (9.9 ml, 57.0mmol). The resulting solution was stirred at room temperature until allstarting material was consumed as determined by LCMS (4 h). The solutionwas concentrated and purified by flash chromatography eluted with 20% to80% ethyl acetate/dichloromethane. Yield 8.8 g, 59.0% for four steps,Ion(s) found by LCMS: M+H 587.3.

Step e

A reaction flask containing product from the previous step (6.5 g, 11mmol) was vacuum flushed with nitrogen and dissolved in anhydrousdichloromethane (100 ml). After the solution was cooled in an ice-waterbath, DMAP (4.89 g, 40 mmol) was added and stirred to dissolve, then4-nitrophenylchloroformate (5.58 g, 28 mmol) was added in portions,while stirring at 0° C. to room temperature for 1 hour. LCMS showsstarting material 1 hr so additional DMAP (1.22 g, 10 mmol) and4-nitrophenylchloroformate (1.0 g, 5 mmol) were added. The reaction wascontinued for 4 more hours, then concentrated and purified by flashchromatography eluting with 20% to 80% ethyl acetate/dichloromethane.Yield 5.1 g, 59.9%. Ion found by LCMS: [M+H]⁺=752.2.

Example 110. Synthesis of Int-71

Step a

To a solution of 2-(2-Boc-aminoethoxy) ethanol (6.15 g, 30 mmol) inanhydrous DCM (60 ml) was added DIPEA (7.8 g, 60 mmol) and DMAP (366.6mg, 3 mmol). P-toluenesulfonyl chloride (6.86 g, 36 mmol) was then addedin portions over 30 minutes. After the resulting mixture was stirred for3 days, it was concentrated by rotary evaporation and purified by RPLC(20% to 70% acetonitrile/water). Yield 3.71 g, 34.4%. Ion found by LCMS:[M-Boc+H]⁺=260.

Step b

To a solution of the step-a product (2.1 g, 5.83 mmol) in anhydrous THF(10 ml) was added sodium carbonate (1.24 g, 11.7 mmol) andN-Boc-1,4-diaminobutane (1.32 g, 7 mmol). The resulting mixture washeated at 60° C. for 1 day. The salt was then filtered off, and thefiltrate was concentrated by rotary evaporation. The residue waspurified by RPLC (100 g, 5 to 50% acetonitrile and water). Yield 1.94 g,88.6%. Ion found by LCMS: [M+H]⁺=376.0.

Step c

To a solution of propargyl PEG-4 acid (781 mg, 3 mmol) and HATU (1.14 g,3 mmol) in anhydrous DMF (3 ml) was added DIPEA (390 mg, 3 mmol),followed by the addition of the solution step-b product (940 mg, 2.5mmol) and DIPEA (390 mg, 3 mmol) in anhydrous DMF (3 ml). The reactionmixture was stirred for 30 minutes, then directly purified by RPLC (5%to 80% acetonitrile and water, using 0.1% TFA as modifier). Yield 960.2mg, 65.3%. Ion found by LCMS: [M+H]⁺=618.3, [M-Boc+H]⁺=518.3.

Step d

The step-c product (960.2 mg, 1.63 mmol) was dissolved in anhydrous THF(6 ml). 4N HCl solution in dioxane (4 ml) was added, and the reactionmixture was stirred overnight. It was then concentrated by rotaryevaporation. The residue was extracted with water (3×3 ml) and ethylacetate (10 ml). The combined aqueous layers were lyophilized. Yield 760mg, 95.1%. Ion found by LCMS: [M+H]⁺=418.0.

Step e

To a mixture of the step-d product (556.2 mg, 1.13 mmol) and DIPEA (741mg, 5.7 mmol) in anhydrous DMF (3 ml) was added in portionsp-nitrophenyl carbonate of zanamivir (1.67 g, 2.26 mmol, described inExample 109) over 20 minutes. The reaction was stirred for 1 hour, thendirectly purified by RPLC (30% to 90% acetonitrile/water, using 0.1% TFAas modifier). Yield 1.35 g, 74%. Ion found by LCMS: [(M+2H)/2]⁺=807.9.

Step f

The step-e product (1.35 g, 0.836 mmol) was dissolved in TFA (5 ml). Thereaction was heated at 30° C. for 1 hour, it was directly purified byRPLC (0% to 35% acetonitrile/water, using 0.1% TFA as modifier). Yield1.00 g, 82.9%. Ions found by LCMS: [(M+2H)/2]⁺=607.8.

Step g

The step-f product (1.00 g, 0.693 mmol) was dissolved in MeOH (12 ml),then cooled in an ice-water bath. It was then treated with a solution ofLiOH monohydrate (286 mg, 6.6 mmol) in water (9 ml). The resultingmixture was stirred overnight and then acidified by 4N HCl solution indioxane (2 ml). After organic solvents were removed by rotaryevaporation, the residue was purified by preparative HPLC (Isco ACCQprep, Luna 5 μm C18(2) 100 Å LC column 100 mm×30 mm; Gradient: 0%acetonitrile/water for 2 min, then 0% to 15% acetonitrile/water over 12min, then isocratic at 15% acetonitrile for 10 min, using 0.1% TFA).Yield 275.9 mg, 29.2%. Ions found by LCMS: [(M+2H)/2]⁺=567.8,[(M+3H)/3]⁺=378.9.

Example 111. Synthesis of Conjugate 31

A 15-ml sterile centrifuge tube was charged with sodium ascorbate (68.1mg, 0.344 mmol), THPTA (14.9 mg, 0.0344 mmol), alkyne derivatized smallmolecule (17.5 mg, 0.00953 mmol, described in Example 110) and bufferPBS 7.4 (1 ml). After stirred by vortex to dissolve everything, Peg 4azido Fc (50 mg, 0.0008588 mmol, described in Example 7 SEQ ID No. 18)was added followed by a solution of CuSO4 (2.05 mg, 0.0129 mmol) in PBS(0.5 ml). The mixture was gently rotated for 20 hours then purified byaffinity chromatography over a protein-A column, followed size exclusionchromatography. Maldi TOF analysis of the purified final product gave anaverage mass of 63586 Da (DAR=3.8). Yield 32.8 mg, 66% yield.

Example 112. Synthesis of Int-72

Step a

To a solution of N-Boc-1,4-diaminobutane (1.56 g, 8.28 mmol) inanhydrous DMF (7 ml) was added sodium carbonate (742 mg, 7 mmol) and5-(Boc-amino)-1-pentylbromide (1.73 g, 6.5 mmol). The resulting mixturewas heated at 50° C. for 24 hours. The salt was then filtered off, andthe filtrate was concentrated by rotary evaporation. The residue waspurified by RPLC (5% to 50% acetonitrile/water, using 0.1% TFA asmodifier). Yield 2 g, 63.2%. Ion found by LCMS: [M+H]⁺=374.4.

Step b

To a solution of propargyl PEG-4 acid (1.3 g, 5 mmol) and HATU (2.1 g,5.5 mmol) in anhydrous DMF (6 ml) was added DIPEA (1.3 g, 10 mmol).After 5 minutes, the reaction mixture was added to the step-a product (2g, 4.1 mmol) and stirred for 1 hour. It was then directly purified byRPLC (5% to 80% acetonitrile/water, using 0.1% TFA as modifier). Yield2.34 g, 95%. Ion found by LCMS: [M+H]⁺=616.4, [M-Boc+H]⁺=516.4.

Step c

The step-b product (2.34 g, 3.8 mmol) was dissolved in anhydrous THF (12ml). 4N HCl solution in dioxane (10 ml) was added, and the reactionmixture was stirred overnight. It was then concentrated by rotaryevaporation. The residue was re-dissolved in acetonitrile/water (1:1,˜16 ml), and the solution was lyophilized. The crude product was carriedto the subsequent step without further purification. Yield 1.91 g,quantitative yield. Ion found by LCMS: [M+H]⁺=416.4.

Step d

To a solution of p-nitrophenyl carbonate of zanamivir (2.25 g, 3.05mmol, described in Example 109) in anhydrous DCM (3 ml) was addedin-portions a mixture of the step-c product (610 mg, 1.249 mmol) andDIPEA (1.05 g, 8.1 mmol) in anhydrous DMF (4 ml) over 20 minutes. Afterstirring for 1 hour, the reaction mixture was concentrated and purifiedby RPLC (30% to 80% acetonitrile/water, using 0.1% TFA as modifier).Yield 1.73 g, 85.9%. Ion found by LCMS: [(M+2H)/2]⁺=806.8,[(M-Boc+2H)/2]⁺=756.8.

Step e

The step-d product (1.73 g, 1.073 mmol) was dissolved in TFA (5 ml).After the solution was heated at 30° C. for 3 hours, it was directlypurified by RPLC (0% to 35% acetonitrile/water, using 0.1% TFA asmodifier). Yield 1.176 g, 76.1%. Ions found by LCMS: [(M+2H)/2]⁺=606.8,[(M+3H)/3]⁺=405.

Step f

The step-e product (1.176 g, 0.817 mmol) was dissolved in MeOH (12 ml),and the solution was cooled in an ice-water bath. It was treateddropwise with a solution of LiOH monohydrate (344.4 mg, 8.2 mmol) inwater (9 ml). The resulting mixture was stirred overnight and thenacidified by 4N HCl solution in dioxane (2 ml). After organic solventswere removed by rotary evaporation, the residue was purified bypreparative HPLC (Isco ACCQ prep, Luna 5 μm C18(2) 100 Å LC column 100mm×30 mm; Gradient: 0% acetonitrile/water for 2 min, then 0% to 15%acetonitrile/water over 12 min, then isocratic at 15% acetonitrile for10 min, using 0.1% TFA). Yield 108 mg, 9.7%. Ions found by LCMS:[(M+2H)/2]⁺=566.8, [(M+3H)/3]⁺=378.2.

Example 113. Synthesis of Conjugate 32

A 15-ml sterile centrifuge tube was charged with sodium ascorbate (68.1mg, 0.344 mmol), THPTA (14.9 mg, 0.0344 mmol), product from Example 112,Int-72 (17.5 mg, 0.00953 mmol) and PBS 7.4 (1 ml). After stirring byvortex to dissolve everything, azido Fc (50 mg, 0.0008588 mmol,described in Example 7 with SEQ ID NO: 4) was added followed by asolution of CuSO4 (2.05 mg, 0.0129 mmol) in PBS (0.5 ml). The mixturewas rotated for 20 hours. It was purified by affinity chromatographyover a protein A column, followed size exclusion chromatography. MaldiTOF analysis of the purified final product gave an average mass of63588. Da (DAR=3.8). Yield 30.9 mg, 62% yield.

Example 114. Synthesis of Int-73

Step a

To a solution of the p-nitrophenyl carbonate of zanamivir (698.4 mg,0.95 mmol, described in Example 109) in anhydrous DCM (2 ml) was addedin-portions over 10 minutes a mixture of propargyl diamine centrallinker (209 mg, 0.426 mmol, described in Example 110) and DIPEA (330.8mg, 2.56 mmol) in anhydrous DMF (2 ml). The reaction was stirred at roomtemperature for 1 hour. It was then concentrated and purified by RPLC(30% to 85% acetonitrile/water, no TFA modifier). Yield 531 mg, 69.2%.Ions found by LCMS: [(M+2H)/2]⁺=807.8, [(M-Boc+2H)/2]⁺=757.8.

Step b

The step-b product (531 mg, 0.329 mmol) was dissolved in DCM (1.5 ml)and TFA (1.5 ml), then stirred at 35° C. for 3 hours. It wasconcentrated and purified by RPLC (5% to 30% acetonitrile/water, no TFAmodifier). Yield 387 mg, 97.1%. Ions found by LCMS: [(M+2H)/2]⁺=607.6,[(M+3H)/3]⁺=405.4.

Step c

The step-b product (121.4 mg, 0.1 mmol) was dissolved in 1.0 M NaClsolution (3 ml) and acetonitrile (1 ml). After the solution was cooledto −8° C. (salt/ice bath), 1.0 M NaOH (0.4 ml, 0.4 mmol) was addeddropwise and the reaction was stirred at −14° C. to −8° C. for 6 hours.It was then neutralized with 4N HCl in dioxane solution (100 μl) anddirectly purified by preparative HPLC (Isco ACCQ prep, Luna 5 μm C18(2)100 Å LC column 100 mm×30 mm; Gradient: 0% acetonitrile/water for 2 min,then 0% to 17.8% acetonitrile/water over 12 min, then isocratic at 17.8%acetonitrile for 10 min, using 0.1% TFA). Yield 85.5 mg, 62.8%. Ionsfound by LCMS: [(M+2H)/2]⁺=567.8, [(M+3H)/3]⁺=378.9.

Example 115. Synthesis of Int-74

Step a

To a solution of N-Boc-2-(2-amino-ethoxy)ethylamine (1.2 g, 5 mmol) inanhydrous DMF (5 ml) was added sodium carbonate (691 mg, 5 mmol) andN-Boc-6-bromo-hexylamine (1.4 g, 5 mmol). The resulting mixture washeated at 70° C. for 24 hours. The salt was then filtered off, and thefiltrate was concentrated by rotary evaporation. The residue waspurified by RPLC (5% to 50% acetonitrile/water, using 0.1% TFA asmodifier). Yield 444 mg, 22%. Ion found by LCMS: [M+H]⁺=404.3.

Step b

To a solution of propargyl PEG-4 acid (342.6 mg, 1.32 mmol) and HATU(601.5 mg, 1.58 mmol) in anhydrous DMF (2 ml) was added DIPEA (258 mg, 2mmol). After 5 minutes, the reaction mixture was added into the step-aproduct (444.3 mg, 0.858 mmol) and stirred for 1 hour. It was thendirectly purified by RPLC (5% to 80% acetonitrile/water, using 0.1% TFAas modifier). Yield 297.1 mg, 53.6%. Ion found by LCMS: [M+H]⁺=646.2,[M-Boc+H]⁺=546.2.

Step c

The step-b product (297.1 mg, 0.46 mmol) was dissolved in anhydrous THF(2 ml). 4N HCl solution in dioxane (4 ml) was added, and the reactionmixture was stirred overnight. It was then concentrated by rotaryevaporation and purified by preparative HPLC (5% to 50%acetonitrile/water, using 0.1% TFA as modifier). Yield 239.6 mg, 77.3%.Ion found by LCMS: [M+H]⁺=446.2.

Step d

To a solution of the p-nitrophenyl carbonate of zanamivir (523.8 mg,0.71 mmol, described in Example 2) in anhydrous DCM (2 ml) was added inportions over 10 minutes a mixture of the step-c product (239.6 mg,0.356 mmol) and DIPEA (245.5 mg, 1.9 mmol) in anhydrous DMF (2 ml). Thereaction was stirred at room temperature for 1 hour. It was thenconcentrated and purified by RPLC (30% to 85% acetonitrile/water, no TFAmodifier). Yield 553 mg, 96.5%. Ions found by LCMS: [(M+2H)/2]⁺=821.8,[(M-Boc+2H)/2]⁺=771.8.

Step e

The step-d product (553 mg, 0.343 mmol) was dissolved in DCM (1.5 ml)and TFA (1.5 ml), then stirred at 35° C. for 3 hours. It wasconcentrated and purified by RPLC (5% to 30% acetonitrile/water, no TFAmodifier). Yield 424.6 mg, 99.6%. Ions found by LCMS: [(M+2H)/2]⁺=621.6,[(M+3H)/3]⁺=415.0.

Step f

The step-e product (424.6 mg, 0342 mmol) was dissolved in 1.0 M NaClsolution (4 ml) and acetonitrile (4 ml). After the solution was cooledto −11° C. (salt/ice bath), 1.0 M NaOH (1.53 ml, 1.53 mmol) was addeddropwise and the reaction was stirred at −14° C. to −8° C. for 6 hours.It was then quenched with 4N HCl in dioxane solution (375 μl) anddirectly purified by preparative HPLC (Isco ACCQ prep, Luna 5 μm C18(2)100 Å LC column 100 mm×30 mm; Gradient: 0% acetonitrile/water for 2 min,then 0% to 20.9% acetonitrile/water over 13.6 min, then isocratic at20.9% acetonitrile for 10 min, using 0.1% TFA). Yield 439 mg, 92.3%.Ions found by LCMS: [(M+2H)/2]⁺=581.8, [(M+3H)/3]⁺=388.2.

Example 116. Synthesis of Int-75

Step a

A mixture of tert-butyl (4-oxobutyl) carbamate (850 mg, 4.54 mg) andtert-butyl N-{2-[2-(2-aminoethoxy) ethoxy]ethyl}carbamate (1.99 g, 8mmol) was dissolved in DCM (30 ml) and dried over Na₂SO₄. Afterfiltering and concentrating, the residue was redissolved in anhydrousDCM (20 ml). Acetic acid (641 mg, 11 mmol) was added, followed by sodiumtriacetoxyborohydride (3.4 g, 16 mmol) in portions over 1 hour. Thereaction mixture was stirred overnight, then quenched with AcOH (3 ml)and MeOH (10 ml). The mixture was filtered, and the filtrate wasconcentrated by rotary evaporation and purified by RPLC (5% to 45%acetonitrile and water). Yield 618 mg, 32.5%. Ion found by LCMS:[M+H]⁺=420.4.

Step b

This compound was prepared analogously to the step-b product of Example115. Ions found by LCMS: [M+H]⁺=662.4, [M-Boc+H]+=562.4.

Step c

This compound was prepared analogously to the step-c product of Example115. Ion found by LCMS: [M+H]⁺=462.4.

Step d

This compound was prepared analogously to the step-d product of Example115. Ions found by LCMS: [(M+2H)/2]⁺=829.8, [(M-Boc+2H)/2]⁺=779.8.

Step e

This compound was prepared analogously to the step-e product of Example115. Ions found by LCMS: [(M+2H)/2]⁺=629.8, [(M+3H)/3]⁺=420.2.

Step f

This compound was prepared analogously to the step-f product of Example115. Ions found by LCMS: [(M+2H)/2]⁺=598.8, [(M+3H)/3]⁺=393.6.

Example 117. Synthesis of Int-76

Step a

To a solution of the N-Boc-peg-1 tosylate (1.54 g, 4.28 mmol, describedin Example 110) in anhydrous THF (8 ml) was added tert-butylN-2{2-[2-(2-aminoethoxy) ethoxy]ethyl}carbamate (1.59 g, 6.42 mmol) andsodium carbonate (453.7 mg, 4.28 mmol). The resulting mixture was heatedat 50° C. for 24 hours. The solid was filtered and washed withacetonitrile. The filtrate was concentrated and purified by RPLC (100 g,5 to 90% acetonitrile and water). Yield 1.15 g, 61.7%. Ion found byLCMS: [M+H]⁺=436.4.

Step b

This compound was prepared analogously to the product from step-b ofExample 115. Ions found by LCMS: [M+H]⁺=678.2, [M-Boc+H]⁺=578.2.

Step c

This compound was prepared analogously to the product of step-c ofExample 115. Ion found by LCMS: [M+H]⁺=478.2.

Step d

This compound was prepared analogously to the product from step-d ofExample 115. Ions found by LCMS: [(M+2H)/2]⁺=837.6,[(M-Boc+2H)/2]⁺=767.8.

Step e

This compound was prepared analogously to the product from step-e ofExample 115. Ions found by LCMS: [(M+2H)/2]⁺=637.5, [(M+3H)/3]⁺=425.6.

Step f

This compound was prepared analogously to the product from step-f ofExample 115. Ions found by LCMS: [(M+2H)/2]⁺=597.8, [(M+3H)/3]⁺=399.0.

Example 118 Synthesis of Int-67

Step a

Previously prepared ether zanamivir acid starting material (0.90 g, 1.43mmol, described in Example 22) and N-methyl morpholine (0.23 mL, 2.14mmol) were dissolved in THF (35 mL) and cooled to 0° C. (ice water bath)under an atmosphere of nitrogen. Isobutyl chloroformate (0.24 mL, 1.85mmol, in 2 mL DCM) was added dropwise, by way of syringe over a 5 minuteperiod. The mixture was stirred at 0° C. for 30 minutes then 15 min atambient temperature, and then cooled to 0° C., where sodium borohydride(540 mg, 14.3 mmol, dissolved in 5 mL of methanol) was added, dropwiseover 5 minutes. The reaction was stirred for 15 minutes at which pointall starting material had been consumed (by LC/MS). A few drops (1 mL)of glacial acetic acid was added to acidify the mixture (pH˜5). Themixture was diluted with ethyl acetate and water and extracted intoethyl acetate (3×). The organic layer was washed with brine, and theorganic extracts were dried over sodium sulfate, and concentrated on arotary evaporator. The crude material was purified by silica gelchromatography, dried onto celite first, and then eluted with 0%-10%methanol in DCM over 30 min. Yield 0.66 g, 75%. Ion found by LCMS:[M+H]+=617.2.

Step b

To a stirring mixture of product from the previous step (0.66 g, 1.10mmol), in 20 mL DCM, was added triethylamine (0.30 mL, 1.3 mmol). Themixture was cooled to 0° C. (ice-water bath) under an atmosphere ofnitrogen then treated with mesyl chloride (0.15 g, 1.3 mmol), dropwiseover 5 minutes by way of syringe. The ice bath was removed and thereaction was stirred for 45 minutes. The reaction was quenched withsaturated aqueous sodium bicarbonate, then extracted into DCM (3×). Thecombined organic extracts were washed with brine, dried over sodiumsulfate and concentrated on the rotary evaporator. Yield 0.74 g, 99%.Ion found by LCMS: [M+H]+=695.2. The intermediate was taken to the nextstep without purification.

Step c

Mesylate from the previous step (0.74 g, 1.1 mmol) was stirred in DMF (5mL) at 80° C. with 3 eq of sodium azide (0.21 g, 3.3 mmol) for 5 hours.The mixture was diluted with water, extracted into DCM (3×). Thecombined organic extracts were washed with brine, dried over sodiumsulfate and concentrated. Yield 0.67 g, 95%. Ion found by LCMS:[M+H]+=642.4. The azide was taken to the next step without purification.

Step d

Azide from the previous step (0.67 g, 1.04 mmol) was stirred in methanol(20 mL) in the presence of Lindlar catalyst (300 mg) under 1 atmosphereof hydrogen gas for 12 hours. The mixture was filtered through celiteand concentrated to afford the title compound as a clear oil. The aminewas taken to the next step without purification. Yield 0.37 g, 54%, 3steps. Ion found by LCMS: [M+H]+=616.2.

Step e

EDC (150 mg, 0.78 mmol) was added to a stirring solution of amine fromthe previous step (370 mg, 0.60 mmol), propargyl-peg4-carboxylic acid(188 mg, 0.72 mmol) and triethylamine (0.100 mL, 0.72 mmoL), dissolvedin DMF (4 mL). The reaction was stirred for 2 hours at ambienttemperature then purified directly RPLC (10%-95% acetonitrile/water, nomodifier, 30 minute gradient). The pure fractions were pooled andlyophilized and taken directly to the next step. Yield 490 mg, 80%. Ionfound by LC/MS: [M+H]⁺=858.2

Step f

Product from the previous step (490 mg, 0.57 mmol) was stirred in TFA (4mL) for 45 minutes then concentrated and azeotroped with methanol (3×)on the rotary evaporator. Ion found by LC/MS: [M+H]⁺=658.2. The residuewas stirred in 1/1 methanol/water containing LiOH (43 mg, 1.8 mmol) for30 minutes. The reaction was neutralized with a few drops of glacialacetic acid and the volume was reduced by half on the rotary evaporator.The crude product was purified by semi-preparative HPLC (0%-75%acetonitrile/water, 0.1% TFA, 30 minute gradient). The pure fractionswere pooled and lyophilized to afford the title compound. Yield 105 mg,29%, 3 steps. Ion found by LC/MS: [M+H]⁺=618.2.

Example 119. Synthesis of Int-68

Step a

p-Nitrophenyl chloroformate (0.23 g, 1.14 mmol) was added to a stirringmixture of the primary alcohol (0.47 g, 0.76 mmol described in Example118) and triethylamine (0.21 mL, 1.52 mmol) dissolved in DCM (15 mL,anhydrous). The reaction was stirred for 1 hour then additionaltrimethylamine (0.21 mL) and p-nitrophenyl chloroformate (230 mg) wereadded, and stirring was continued another hour, at which time themixture was diluted with water, and extracted into DCM (3×). Thecombined organic extracts were washed with brine and dried over sodiumsulfate. The solvent was removed by rotary evaporation. The cruderesidue was dissolved in DCM (3 mL), loaded onto celite and purified bysilica gel chromatography (0% to 70% ethyl acetate/hexanes, 30 minutegradient). The pure fractions were pooled and concentrated to afford thetitle compound as a white solid. Yield 525 mg, 88%. Ion found by LCMS:[M+H]⁺=782.2.

Step b

Triethylamine (0.14 mL, 0.97 mmol) was added to a stirring solution ofpropargyl-peg4-amine (181 mg, 0.78 mmol) in 1 mL of acetonitrile. Thetriethylamine/propargyl-peg4-amine mixture was added to the product fromthe previous step (510 mg, 0.65 mmol, in 2 mL acetonitrile) and stirredfor 1 hour at which point all starting materials had been consumed. Thesolvent was removed on a rotary evaporator. The crude residue waspurified RPLC (20-95% acetonitrile/water, 30 min gradient, no modifier).Yield 475 mg, 83%. Ion found by LC/MS: [M+H]⁺=874.2.

Step c

The product from the previous step (475 mg, 0.54 mmol), was stirred inTFA (5 mL) for 2 hours and then concentrated and azeotroped withmethanol (3×) on a rotary evaporator. Ion found by LC/MS: [M+H]⁺=674.2.The residue was stirred in 1:1 mixture of methanol/water containing LiOH(49 mg, 1.6 mmol) for 30 minutes. The reaction was neutralized withglacial acetic acid and then concentrated by rotary evaporation. Theproduct was purified by semi-preparative HPLC (0%-75%acetonitrile/water, 0.1% TFA, 30 minute gradient). The pure fractionswere pooled and lyophilized. Yield 204 mg, 59%. Ion found by LCMS:[M+H]⁺=634.2.

Example 120 Synthesis of Int-77

Step a

HATU (0.44 g, 1.16 mmol, in 1.5 mL of DMF) was added dropwise to astirring solution of propargyl-peg4-amine (0.24 g, 1.05 mmol),bis-Boc-D-ornithine (0.35 g, 1.05 mmol), and triethylamine (0.59 mL,4.21 mmol) in DMF (2 mL). The reaction was stirred for 1 hour at ambienttemperature, then purified directly by RPLC (5%-90% acetonitrile/water,0.1% TFA, 35 minute gradient). The pure fractions were pooled andlyophilized to afford the product as a viscous clear oil. Ion found byLC/MS: [M+H]⁺=546.2

The Boc-protected intermediate was stirred in 4N HCL in dioxane (10 mL)for 45 minutes at ambient temperature. The solvent was removed by rotaryevaporation, then the resulting residue was dissolved in DI water (20mL), frozen, and lyophilized to afford the product as a clear oil. Yield310 mg, 70%, 2 steps. Ion found by LCMS: [M+H]⁺=346.2

Step b

The product from the previous step (96 mg, 0.28 mmol) and triethylamine(0.15 mL, 1.11 mmol) in acetonitrile (2 mL) were added to a stirringsolution of p-nitrophenyl carbonate of zanamivir (435 mg, 0.56 mmol,described in Example 118, in 6 mL of acetonitrile), and stirred for 12hours at ambient temperature, then 2 hours at 80° C. The solvent wasremoved and the crude residue was purified by RPLC (10-95%acetonitrile/water, no modifier, 35 minute gradient). The pure fractionswere pooled and concentrated on a rotary evaporator. Ion found by LC/MS:[(M+2H)/2]⁺=815.8

This intermediate was stirred in TFA (5 mL) for 45 minutes thenconcentrated and dried under high vacuum. Ion found byLC/MS[(M+2H)/2]⁺=615.8.

This TFA salt was stirred in (1/1) MeOH/DI water (5 mL) containing LiOH(27 mg, 1.11 mmol) for 30 minutes at 0° C. The mixture was acidified(˜pH˜5) with glacial acetic acid. The methanol was removed by rotaryevaporation and the resulting residue was purified by semi-preparativeHPLC (5%-70% acetonitrile/water, 0.1% TFA, 35 minute gradient). The purefractions were pooled and lyophilized. Yield 35 mg, 11%, 3 steps. Ionfound by LC/MS: [(M+2H)/2]⁺=575.8.

Example 121. Synthesis of Int-78

Step a

Propargyl-Peg4-acid (640 mg, 2.46 mmol), diol-HCl salt (350 mg, 2.46mmol), EDC (471 mg, 2.46 mmol), HOBt (377 mg, 2.46 mmol), andtriethylamine (249 mg, 2.46 mmol) were stirred in DMF (3 mL) at ambienttemperature for 4 hours. The mixture purified directly by RPLC (0%-80%acetonitrile/water, no modifier, 35 minute gradient). The pure fractionswere pooled and concentrated to afford the product as a clear oil. Yield580 mg, 68%. Ion found by LCMS: [M+H]⁺=348.4.

Step b

p-Nitro-phenyl chloroformate (1.23 g, 1.25 mmol) was added to a stirringsolution of product from the previous step (530 mg, 6.10 mmol) andtriethylamine (925 mg, 9.15 mmol) in DCM (25 mL), then cooled to 0° C.under a nitrogen atmosphere. The mixture was stirred for 15 minutes at0° C. and then at room temperature for 2 hours. The reaction was dilutedwith DI water and extracted into DCM (3×20 mL). The combined organicextracts were washed with brine and dried over sodium sulfate. The cruderesidue was purified by silica gel chromatography (10%-100% ethylacetate/hexanes, 25 minute gradient) to afford the product as a whitesolid. Yield, 250 mg, 24%. Ion found by LCMS: [M+H]⁺=678.2.

Step c

Amine functionalized zanamivir (505 mg, 0.82 mmol, described in Example118) and triethylamine (0.15 mL, 1.11 mmol) in acetonitrile (2 mL) wereadded to a stirring solution of the product of the previous step (220mg, 0.37 mmol) in acetonitrile (10 mL), and stirred for 4 hours atambient temperature. The reaction was concentrated on the rotaryevaporator. The resulting residue was purified by RPLC (15%-95%acetonitrile/water, no modifier, 35 minute gradient). The pure fractionswere pooled and concentrated on a rotary evaporator. Ion found by LC/MS:[(M+2H)/2]⁺=815.2.

This intermediate was stirred in TFA (5 mL) for 45 minutes thenconcentrated and dried under high vac. Ion found by LC/MS:[(M+2H)/2]⁺=615.2.

The resulting TFA salt was stirred in (1/1) MeOH/DI water (5 mL)containing LiOH (71 mg, 2.98 mmol) for 30 minutes at 0° C. The mixturewas acidified (˜pH˜5) with glacial acetic acid. The methanol was removedby rotary evaporator and purified by semi-preparative HPLC (5%-70%acetonitrile/water, 0.1% TFA, 35 minute gradient). The pure fractionswere pooled and lyophilized. Yield 125 mg, 29% for 3 steps. Ion found byLC/MS: [(M+2H)/2]⁺=575.8.

Example 122. Synthesis of Int-4a

Step a

Propargyl-Peg 4-amine (165 mg, 0.71 mmol) was added to a stirringsolution of p-nitrophenyl carbonate of zanamivir (350 mg, 0.47 mmol,described in Example 109) in acetonitrile (20 mL). The reaction wasstirred for 1 hour at ambient temperature, then solvent was removed byrotary evaporator. The residue was purified by RPLC (10%-95%acetonitrile/water, no modifier, 30 minute gradient). The pure fractionswere pooled and lyophilized to afford the boc protected intermediate asa white solid. Ion found by LCMS: [M+H]⁺=830.2.

This intermediate was stirred in TFA (5 mL) at ambient temperature for30 minutes. The solvent was removed by rotary evaporator and the residuewas purified by RPLC (10%-95% acetonitrile/water, 0.1% TFA, 30 minutegradient). The pure fractions were pooled and lyophilized to afford theproduct as a white solid. Yield 155 mg, 52%, 2 steps. Ion found by LCMS:[M+H]⁺=630.2.

Step b

The product from the previous step (140 mg, 0.22 mmol) was stirred in a1:3 mixture of methanol:water (8 mL) containing LiOH (21 mg, 0.89 mmol)at 0° C. for 20 minutes. The mixture was acidified with a few drops ofglacial acetic acid and concentrated on a rotary evaporator. The crudematerial was purified by semi-preparative HPLC (5%-95%acetonitrile/water, 0.1% TFA, 30 minute gradient). The pure fractionswere pooled and lyophilized to afford the product as a white solid.Yield 60 mg, 45%. Ion found by LCMS: [M+H]⁺=590.2.

¹H NMR (500 MHz, Methanol-d₄) δ 5.86 (d, 0=2.6 Hz, 1H), 4.99 (dd, J=9.0,2.7 Hz, 1H), 4.55 (dd, J=9.7, 2.7 Hz, 1H), 4.38 (dd, J=8.6, 2.6 Hz, 1H),4.24-4.14 (m, 1H), 4.19 (d, J=2.6 Hz, 2H), 4.02-3.98 (m, 1H), 3.74-3.59(m, 13H), 3.54 (t, J=5.6 Hz, 2H), 3.52-3.48 (m, 1H), 3.29-3.22 (m, 2H),2.84 (t, J=2.4 Hz, 1H), 1.96 (s, 3H).

¹³C NMR (126 MHz, MeOD) δ 172.33, 163.68, 157.54, 156.58, 106.80, 79.23,75.91, 74.56, 70.18, 70.13, 69.96, 69.87, 69.59, 69.51, 69.14, 68.74,62.98, 57.67, 51.11, 40.54, 21.37.

Example 123. Synthesis of Int-4b

Peg functionalized intermediate (100 mg, 0.13 mmol, described in Example122) was stirred in a 1:3 mixture of methanol:water (5 mL) containingLiOH (12 mg, 0.52 mmol) at ambient temperature for 2 hours. The mixturewas acidified with glacial acetic acid and concentrated by rotaryevaporator. The crude material was purified by RPLC (5-95% acetonitrilein DI water, 0.1% TFA, 30 minute gradient). The pure fractions werepooled and lyophilized to afford the product as a white solid. Yield 21mg, 31%. Ion found by LCMS: [M+H]⁺=590.2.

¹H NMR (Methanol-d₄) δ: 5.86 (d, J=2.6 Hz, 1H), 4.48 (dd, J=8.6, 2.7 Hz,1H), 4.42 (dd, J=9.6, 1.4 Hz, 1H), 4.38 (d, J=12 Hz, 1H), 4.22-4.19 (m,1H), 4.19 (d, J=2.4 Hz, 2H), 4.16-4.12 (dd, J=11.5, 6 Hz, 1H), 4.09-4.02(m, 1H), 3.75-3.58 (m, 13H), 3.55 (t, J=5.5 Hz, 2H), 3.33-3.29 (m, 2H),2.84 (t, J=2.4 Hz, 1H), 2.02 (s, 3H).

¹³C NMR (126 MHz, MeOD) δ 172.94, 163.82, 157.90, 157.53, 106.80, 79.23,76.18, 74.55, 70.19, 70.13, 69.96, 69.90, 69.61, 68.96, 68.74, 68.17,66.69, 57.67, 50.11, 40.43, 21.33.

Example 124. General Procedure for Synthesis of Azido Fc

Preparation of PEG4-azido NHS ester solution (0.050 M) in DMF/PBS: 16.75mg of PEG4-azido NHS ester was dissolved in 0.100 mL of DMF at 0° C. anddiluted to 0.837 mL by adding PBS 1× buffer at 0° C. This solution wasused for preparing other PEG4-azido Fc with a variety of DAR values byadjusting the equivalents of this PEG4-azido NHS ester PBS solution.

Pretreatment of h-IgG1 Fc, SEQ ID NO: 48 (107.2 mg in 8.800 mL of pH 7.4PBS, MW-57891 Da, 1.852 μmol): The Fc solution was transferred into fourcentrifugal concentrators (30,000 MWCO, 15 mL) and diluted to 15 mL withPBS×1 buffer and concentrated to a volume of 1.5 mL. The residue wasdiluted 1:10 in PBS pH 7.4, and concentrated again. This wash procedurewas repeated for total of four times followed by dilution to 8.80 mL.

Preparation of PEG4-azido Fc: 0.050M PEG4-azidoNHS ester PBS buffersolution (0.593 mL, 29.6 μmol, 16 equivalents) was added to abovesolution of h-IgG1 Fc (SEQ ID NO: 48) and the mixture was shaken rotatedfor 2 hours at ambient temperature. The solution was concentrated byusing four centrifugal concentrators (30,000 MWCO, 15 mL) to a volume of1.5 mL. The crude mixture was diluted 1:10 in PBS pH 7.4, andconcentrated again. This wash procedure was repeated for total of threetimes. The concentrated Fc-PEG4-azide was diluted to 8.80 mL with pH 7.4PBS buffer and ready for Click conjugation. The purified material wasquantified using a NANODROP™ UV visible spectrophotomer (using acalculated extinction coefficient based on the amino acid sequence ofh-IgG1). Yield was quantitative after purification.

Example 125. Synthesis of Conjugate 34

A solution of azido functionalized Fc (40 mg, 2.5 mL, 0.69 μmol,described in the general preparation of azido Fc, Example 124, SEQ IDNo. 18) was added to a 15 mL centrifuge tube containing alkynederivatized small molecule (6.0 mg, 8.23 μmol, Int-67 Example 118).After gently agitating to dissolve all solids, the mixture was added toa solution of L-ascorbic acid sodium salt (54 mg, 0.27 mmol), copper(II) sulfate (0.88 mg, 5.5 μmol), and BTTA (9.4 mg, 22 μmol) in PBS 7.4buffer (1.09 mL). The resulting mixture was gently rotated overnight. Itwas then purified by affinity chromatography over a protein A column,followed size exclusion chromatography (See general conjugatepurification protocol). Maldi TOF analysis of the purified final productgave an average mass of 64,678 Da (DAR=7.2). Yield 24 mg, 54% yield.

Example 126. Synthesis of Conjugate 35

A solution of azido functionalized Fc (40 mg, 2.5 mL, 0.69 μmol,described in the general preparation of azido Fc, Example 124, SEQ IDNo. 18) was added to a 15 mL centrifuge tube containing alkynederivatized small molecule (6.1 mg, 8.23 μmol, Int-68 Example 119).After gently agitating to dissolve all solids, the mixture was added tosolution of L-ascorbic acid sodium salt (54 mg, 0.27 mmol), copper (II)sulfate (0.88 mg, 5.5 μmol), and BTTA (9.4 mg, 22 μmol) in PBS 7.4buffer (1.09 mL). The resulting solution was gently rotated overnight.It was then purified by affinity chromatography over a protein A column,followed size exclusion chromatography (See general conjugatepurification protocol). Maldi TOF analysis of the purified final productgave an average mass of 64,830 Da (DAR=7.3). Yield 23 mg, 52% yield.

Example 127. Synthesis of Conjugate 36

A solution of azido functionalized Fc (50 mg, 2.5 mL, 0.86 μmol,described in the general preparation of azido Fc, Example 124, SEQ IDNo. 18) was added to a 15 mL centrifuge tube containing alkynederivatized small molecule (7.1 mg, 5.2 μmol, Int-77, Example 120).After gently agitating to dissolve all solids, the mixture was added toa solution of L-ascorbic acid sodium salt (68 mg, 0.34 mmol), copper(II) sulfate (1.1 mg, 6.9 μmol), and BTTA (12 mg, 27 μmol) in PBS 7.4buffer (1.37 mL). The resulting mixture was gently rotated overnight. Itwas then purified by affinity chromatography over a protein A column,followed size exclusion chromatography (See general conjugatepurification protocol). Maldi TOF analysis of the purified final productgave an average mass of 63,300 Da (DAR=3.6). Yield 37 mg, 73% yield.

Example 128. Synthesis of Conjugate 37

A solution of azido functionalized Fc (70 mg, 2.5 mL, 1.2 μmol,described in the general preparation of azido Fc, Example 124, SEQ IDNo. 18) was added to a 15 mL centrifuge tube containing alkynederivatized small molecule (13.3 mg, 9.6 μmol, Int 78, Example 121).After gently agitating to dissolve all solids, the mixture was added toa solution of L-ascorbic acid sodium salt (96 mg, 0.48 mmol), copper(II) sulfate (1.6 mg, 10 μmol), and BTTA (17 mg, 38 μmol) in PBS 7.4buffer (1.92 mL). The resulting mixture was gently shaken overnight. Itwas then purified by affinity chromatography over a protein A column,followed size exclusion chromatography (See general conjugatepurification protocol). Maldi TOF analysis of the purified final productgave an average mass of 63,574 Da (DAR=3.6). Yield 42 mg, 61% yield.

Example 129. Synthesis of Conjugate 33

Preparation of the Click reagent solution: 0.0050M CuSO₄ in PBS buffersolution: 10.0 mg CuSO₄ was dissolved in 12.53 mL PBS, then took 5.00 mLthis CuSO₄ solution and added 43.1 mg BTTAA (CAS #1334179-85-9) and247.5 mg sodium ascorbate to give the Click reagent solution (0.0050MCuSO4, 0.020M BTTAA and 0.25M sodium ascorbate).

To a solution of azido functionalized Fc (104.9 mg, 8.60 mL, 18.1 μmol;Example 124, SEQ ID NO: 64) in a 15 mL centrifuge tube was added toalkyne derivatized small molecule (29.7 mg, 19.9 μmol, described inExample 100, 2.5 equivalents for each azido on the Fc). After gentlyagitating to dissolve all solids, the mixture was treated with the Clickreagent solution (4.34 mL) of (L-ascorbic acid sodium, 0.25 M, 1086μmol, copper (II) sulfate 0.0050M, 21.7 μmol, and BTTAA 0.020M, 86.9μmol). The resulting mixture was gently rotated for 6 hours at ambienttemperature. It was purified by affinity chromatography over a protein Acolumn, followed size exclusion chromatography (see general conjugatepurification protocol). Maldi TOF analysis of the purified final productgave an average mass of 64550 Da (DAR=4.6). Yield 90.7 mg with 98%purity.

Example 130. Efficacy of Conjugate 33 Against InfluenzaB/Brisbane/60/2008 in a Lethal Mouse Model

Test articles were evaluated against a lethal Influenza B influenzainfection in female BALB/c mice (Jackson Laboratories, 6-8 weeks old).The challenge virus (B/Brisbane/60/2008) is a mouse-adapted isolatecapable of causing lethal infections in mice. The experiment comprised10 groups of 5 mice each. At day 0, all mice were challenged with virusat 3× the LD₉₅ by intranasal inoculation in a volume of 50 μl (approx.1E5 virus per mouse), after being anesthetized with isoflurane.

All groups received a single IV treatment of conjugate 33 (Example 129),vehicle (PBS), or Fc only control (hIgG1 Fc) two hours post viralchallenge. An additional group was treated orally with oseltamivir (20mg/kg, bid, for 5 days) starting 8 hours after viral challenge.

The study evaluated 7 different dose concentrations of conjugate 33 (10,3, 1, 0.3, 0.1, 0.03, and 0.01 mg/kg). Mice were monitored for 2 weeksand animals exceeding 20% body weight loss, or were found moribund, werescored as a mortality. Body weights were also recorded to monitor thegeneral health of the animals.

All mice treated with vehicle, or the Fc only control, reached mortalityby Day 8 as expected. In contrast, all mice receiving conjugate 33 werefully protected after receiving single IV doses from 10 down to 0.3mg/kg for the duration of the study (Table 48). In contrast, theoseltamivir treated group only resulted in 40% survival although thecumulative dose these mice received was 200 mg/kg over the course of theexperiment. The potency of conjugate 33 was further supported by dailybody weight measurements (Table 49; data only shown until the firstdeath within a group). Mice treated with conjugate 33 at 0.3 mg/kg onlydemonstrated a transient loss of body weight reaching a maximum of 7%,for a single day. Collectively this study demonstrates the potency ofconjugate 33 as measured by two parameters indicative of Influenza Binfection (survival and body weight) against an influenza strain of theYamagata lineage.

TABLE 48 % survival Fc Conjugate 33 Day (10 (10 to 0.1 post- mg/ 0.3(mg/ (0.03 (0.01 infection PBS kg) mg/kg) kg) mg/kg) mg/kg) Oseltamivir0 100 100 100 100 100 100 100 1 100 100 100 100 100 100 100 2 100 100100 100 100 100 100 3 100 100 100 100 100 100 100 4 60 100 100 100 100100 100 5 40 40 100 100 60 80 40 6 0 0 100 60 60 40 0 7 0 0 100 60 60 00 8 0 0 100 60 60 0 0 9 0 0 100 60 60 0 0 10 0 0 100 60 60 0 0 11 0 0100 60 60 0 0 12 0 0 100 60 60 0 0 13 0 0 100 60 60 0 0 14 0 0 100 60 600 0

TABLE 49 Body weights (gm) Fc Conjugate 33 Day (10 (10 (3 (1 (0.3 (0.1(0.03 (0.01 post- mg/ mg/ mg/ mg/ mg/ mg/ mg/ mg/ infection PBS kg) kg)kg) kg) kg) kg) kg) kg) Oseltamivir 0 100 100 100 100 100 100 100 100100 100 1 99 101 103 98 98 98 98 99 98 98 2 95 97 100 99 97 101 97 97 9797 3 87 90 101 98 96 97 93 95 92 89 4 82 84 104 99 99 97 93 93 88 85 578 79 102 99 99 97 92 84 79 6 98 96 98 93 79 7 102 98 100 96 8 101 98 9897 9 104 101 102 101 10 103 100 101 101 11 104 102 101 101 12 104 101102 102 13 103 102 102 102 14 103 102 102 101

Example 131. Characterization of Regioisomers

Monomer and dimer intermediates may be produced as a particularregioisomer or a mixture of regioisomers. Examples 100-102 showed how toprepare Int-7 regioisomers (C7-C7, Example 100; C7-C9, Example 101;C9-C9, Example 102; C7-C7 optimized, Example 103). The methods describedtherein can be used to separate mixtures of regioisomers of anyintermediate described herein. Table 50 provides the characterization ofthe relative percent (%) amounts of C7 and C9 linked monomers and C7-C7,C7-C9, and C9-C9 linked dimers in the previously described syntheses ofthe pre-conjugation intermediate.

TABLE 50 Regiosomer Analysis Monomer (M) or Int Example Dimer (D) % C7 %C7-C9 % C9 Int-2 Example 6 D 95 5 Int-3 Example 11 D 70 30 Int-4 Example13 M 58 42 Int-4a Example 122 M 97 Int-7 Example 19 D 3 2 95 Int-7aExample 100 D 100 Int-7b Example 101 D 5 95 Int-7c Example 102 D 100Int-25 N/A D 5 95 Int-26 N/A D 25 50 25 Int-27 N/A D 29 35 36 Int-28 N/AD 5 28 67 Int-31 N/A D 90 Int-34 N/A D 12 6 82 Int-37 N/A M 95 Int-71Example 110 D 3 97 Int-72 Example 112 D 100

Example 132. Efficacy of Conjugate 33 Subcutaneously Dosed AgainstInfluenza A/Puerto Rico/8/34 (H1N1) in a Lethal Mouse Model

Conjugate 33 was evaluated against a lethal IAV H1N1 influenza infectionin female BALB/c mice (Charles River Laboratories, 6-8 weeks). Thechallenge virus (A/Puerto Rico/8/1934) is a mouse-adapted isolatecapable of causing lethal infections in mice. The experiment comprised 6groups of 5 mice. At day 0, all mice were challenged with virus at 3×the LD₉₅ by intranasal inoculation in a volume of 30 μl, after beinganesthetized with a mixture of ketamine/xylazine (150 and 10 mg/kgrespectively). Mortality and body weights were recorded daily and anyanimal with a 20% loss of body weight was scored as a death. Test groupsreceived a single subcutaneous (SC) treatment of conjugate 33, hIgG1 Fccontrol, or vehicle (PBS) 2 hours post viral challenge. The study designis summarized in Table 51.

TABLE 51 Study design for Influenza A/PR/8/34 (H1N1) study Dose Dosevolume Group Test article Route/Schedule (mg/kg) (ml/kg) # of mice 1Vehicle SC, T + 2 hrs. na 10 5 2 hIgG1 Fc SC, T + 2 hrs. 1 10 5 3Conjugate 33 SC, T + 2 hrs. 1 10 5 4 Conjugate 33 SC, T + 2 hrs. 0.3 105 5 Conjugate 33 SC, T + 2 hrs. 0.1 10 5 6 Conjugate 33 SC, T + 2 hrs.0.03 10 5

As expected, mice receiving vehicle or the hIgG1 Fc control succumbed toinfection on Days 6-7 (Table 52). However, mice treated with conjugate33 were fully protected at 1, 0.3, and 0.1 mg/kg dose levels. Mortalitywith conjugate 33 was only seen at the lowest dose concentration of 0.03mg/kg.

TABLE 52 Percent survival by study day (n = 5) hIgG1 Fc Conjugate 33 DayPost (1.0 (1.0 (0.3 (0.1 (0.03 Infection Vehicle mg/kg) mg/kg) mg/kg)mg/kg) mg/kg) 0 100 100 100 100 100 100 1 100 100 100 100 100 100 2 100100 100 100 100 100 3 100 100 100 100 100 100 4 100 100 100 100 100 1005 100 100 100 100 100 100 6 40 60 100 100 100 100 7 0 0 100 100 100 60 80 0 100 100 100 0 9 0 0 100 100 100 0 10 0 0 100 100 100 0 11 0 0 100100 100 0 12 0 0 100 100 100 0 13 0 0 100 100 100 0 14 0 0 100 100 100 0

The potency of conjugate 33 was further supported by daily body weightmeasurements. As expected, mice treated with vehicle or hIgG1 Fcdemonstrated a steady drop in body weight until it exceeded 20%, atwhich time they were scored as a mortality (Table 53).

In contrast to control mice, those groups receiving conjugate 33 at 1,0.3, and 0.1 mg/kg maintained healthy body weights throughout the studyand never demonstrated more than a transient body weight drop of lessthan 8% (0.1 mk/kg dose group, Day 8; Table 53). By both survival andbody weight measurements conjugate 33 demonstrated robust protectionfrom a lethal challenge of Influenza A/Puerto Rico/8/1934 with a singleSC dose as low as 0.1 mg/kg.

TABLE 53 Body weight data (gm) hIgG1 Fc Conjugate 33 Day Post (1.0 (1.0(0.3 (0.1 (0.03 Infection Vehicle mg/kg) mg/kg) mg/kg) mg/kg) mg/kg) 0100 100 100 100 100 100 1 98.1 97.6 98 98.6 98.7 98.5 2 100.9 99 100.3101.3 101.6 99.9 3 94.1 92.6 98.4 100.5 96.5 93.9 4 86 85.7 102.6 99.293.5 88.6 5 79.8 79.1 98.2 97.7 94.9 86.5 6 75.3 75.2 102.2 102.3 99.484.6 7 103.5 102.9 95.2 78.4 8 101.8 103.2 92.4 9 101.2 102.9 95.4 10101.3 102.5 98.8 11 102.8 102.6 98.7 12 102.4 100.7 98.6 13 102.2 101.198.3 14 100.2 102 99.6

Example 133. Efficacy of Conjugate 33 Against A/PR/8/1934 (H1N1), LungPFU Burden and Cytokine Levels

Efficacy studies for conjugate 33 were conducted in 6-8 weeks femaleBALB/c mice (Charles River) challenged intranasally with 3E2 PFU/mouse(3× the LD₉₅) of mouse-adapted influenza A/PR/8/1934 (H1N1). Conjugate33 or human IgG1 Fc controls was administered as a single subcutaneously(SC) dose 2 h post-challenge at 0.1-3 mg/kg. Oseltamivir was dosedorally, twice daily for 4 days starting 2 h post-infection at 5 or 50mg/kg. Baloxavirwas resuspended in 0.5% methylcellulose and dosedorally, twice daily for 4 days starting 2 h post-infection at 30 mg/kg(Table 54). Body weights (BW) were recorded for 4 days (FIGS. 62A-62B.At 4 days post-infection, mice were sacrificed by CO₂ and both lunglobes were harvested. Lungs were homogenized with 1 mm silica beads in 1mL PBS using a MagNA Lyser (Roche). Homogenization was carried out at6,000 rpm for 60 s and chilled on ice for 5 min in-between runs. Afterlung homogenization tubes were centrifuged for 10 min at 600×g andsupernatant was transferred into new tube.

TABLE 54 Study Design Test Article (DAR) Route/Sechdule Dose [mg/kg]hIgG1 Fc IV, T + 2 h 3 Oseltamivir PO, BID × 4 5 Oseltamivir PO, BID × 450 Baloxavir SC, BID × 4 30 Conjugate 33 (4.7) SC, T + 2 h 0.1 Conjugate33 (4.7) SC, T + 2 h 0.3 Conjugate 33 (4.7) SC, T + 2 h 1 Conjugate 33(4.7) SC, T + 2 h 3 uninfected N/A N/A

For PFU determination, supernatants of lung homogenate were diluted ininfection buffer ranging from 10⁻¹ to 10⁻⁶. 100 μL of virus dilutionswere added to confluent monolayer of MDCK cells in 24 well plates andincubated for 1 h at room temperature with rocking every 15 min. Afterremoving the virus, liquid overlay media containing Avicel was added toMDCK cells. Cells were incubation at 37° C. 5% CO₂ for 40 h. Afterincubation, the media was removed and cells were stained with crystalviolet to enumerate plaques and Plaque forming units (PFU) werecalculated relative to weight of the lung (PFU/g lung).

For cytokine analysis, supernatants of lung homogenate were seriallydiluted 2-fold in 96 well plate. Cytokine levels for INF-γ, TNF-α, IL-6,MIP-1α and MCP-1 were determined by ELISA according to manufacturer'sinstructions (R&D Systems). After a lethal challenge with influenza in amouse model, lung PFU burden (FIGS. 63A-63B) and lung cytokine levelswere determined on day 4 post-infection (Table 55, and Table 56,respectively). Conjugate 33 demonstrated a dose-dependent log reductionin viral burden resulting in 0.7 at 0.1 mg/kg, 1.88 at 0.3 mg/kg, 3 at 1mg/kg and 3.8 at 3 mg/kg. Oseltamivir at 5 mg/kg and 50 mg/kg had modesteffects on viral burden resulting in 0.86 and 2 log reduction,respectively. Baloxavir reduced the viral burden below the limit ofdetection, 1e2 PFU/mL, thereby reducing the viral burden by >5.99 logsas compared to PBS control.

No biological relevant difference was observed between negativecontrols, PBS and hIgG1 Fc as expected.

Conjugate 33 reduces viral burden in dose dependency on day 4post-infection challenged with influenza A in a mouse model (Table 55,FIGS. 64A-64B). Similarly, conjugate 33 demonstrated a dose-dependentfold-reduction in cytokine levels for TNF-α, IL-6, INF-γ, MCP-1, andMIP-1α (FIGS. 65A-65E, respectively) compared to uninfected control onday 4 post-infection challenged with influenza A in a mouse model (Table56).

TABLE 55 PFU burden Test article [mg/kg] PFU/g Log reduction PBS [0]PBS3.26E+07 0.0 hIgG1 Fc [3] 1.74E+07 0.364 Oseltamivir [5] 4.78E+06 0.86Oseltamivir [50] 3.81E+05 2 Baloxavir [30] <1.00E+02* >5.99* AVC-076-5B[0.1] 6.85E+06 0.715 AVC-076-5B [0.3] 5.31E+05 1.88 AVC-076-5B [1]3.69E+04 2.99 AVC-076-5B [3] 5.84E+03 3.77 *Baloxavir reduced the viralburden below the limit of detection.

TABLE 56 Lung cytokine levels Test article [mg/kg] INF-γ TNF-α IL-6MCP-1 MIP-1α PBS [0] 1.6 1.4 4.2 38.6 18.1 hIgG1 Fc [3] 1.7 1.2 3.3 36.315.9 Oseltamivir [5] 1.0 0.8 1.8 24.0 6.3 Oseltamivir [50] 0.8 0.8 1.416.2 4.0 Baloxavir [30] 1.2 1.0 1.0 1.0 0.8 conjugate 33 [0.1] 1.1 0.70.8 19.1 5.6 conjugate 33 [0.3] 1.1 0.9 0.8 9.6 3.9 conjugate 33 [1] 0.90.7 1.1 5.7 1.7 conjugate 33 [3] 0.8 0.7 1.1 1.8 1.0 Uninfected 1 1 1 11

The highest concentration tested of conjugate 33 at 3 mg/kg demonstratedno weight loss throughout the course of infection similar to uninfectedcontrol mice (Table 57).

TABLE 57 Body weight data (% reduction) Test article [mg/kg] Day 0 Day 1Day 2 Day 3 Day 4 PBS [0] 0 −3.5 −2.86 −10.96 −17.32 hIgG1 Fc [3] 0 −2.6−1.3 −9.58 −15.6 Oseltamivir [5] 0 −2.86 −2.42 −8.16 −13.88 Oseltamivir[50] 0 −3.78 −2.12 −3.22 −5.74 Baloxavir [30] 0 −2.38 −2.82 −2.6 −0.18conjugate 33 [0.1] 0 −3.24 −2.32 −10.12 −11.46 conjugate 33 [0.3] 0−2.16 1.34 −3.4 −4.36 conjugate 33 [1] 0 −1.5 −0.9 −1.98 −0.9 conjugate33 [3] 0 −1.7 −1.6 −1.9 0.52 Uninfected 0 1.02 −0.14 −0.16 2.8

Example 134. Efficacy of Conjugate 33 Against Influenza A (H1N1) in aLethal Severe Combined Immunodeficiency (SCID) Mouse Model

Test articles were evaluated against a lethal Influenza A influenzainfection in female BALB/c scid mice (Jackson Laboratories, 6-8 weeksold). The challenge virus (A/Puerto Rico/8/1934) is a mouse-adaptedisolate capable of causing lethal infections in mice. The experimentcomprised 11 groups of 5 mice each. At day 0, all mice were challengedwith virus at 3× the LD₉₅ by intranasal inoculation in a volume of 30 μl(approx. 1E3 virus per mouse), after being anesthetized with a mixtureof ketamine/xylazine (150 and 10 mg/kg respectively).

Groups received a single SC treatment of vehicle (PBS), hIgG1 Fccontrol, or conjugate 33 (3, 1, 0.3, 0.1, 0.03 mg/kg) two hours postviral challenge. A separate arm of the study consisted of 3 groups ofmice treated with baloxavir marboxil (DC Chemicals, Shanghai, China)orally, twice daily, for 1 day; also starting 2 hours post viralchallenge. Mice were monitored for 2 weeks and animals exceeding 20%body weight loss, or were found moribund, were scored as a mortality.

At study end (Day 14) mice receiving conjugate 33 were fully protectedat all dose concentrations between 3 and 0.1 mg/kg (Table 58). Conjugate33 only failed to protect against lethal viral challenge at the lowesttested concentration of 0.03 mg/kg. As expected, groups receivingvehicle or hIgG1 Fc were not protected. Mice treated with baloxavir werealso protected, but at the significantly higher cumulative doses of 60and 20 mg/kg, at a total dose of 6 mg/kg only 40% of mice survived toDay 14.

TABLE 58 % Survival on Day 14. hIgG1 Fc Baloxavir Conjugate 33 (3.0 (30(10 (3.0 (3.0 (1.0 (0.3 (0.1 (0.03 Vehicle mg/ mg/ mg/ mg/ mg/ mg/ mg/mg/ mg/ (PBS) kg) kg) kg) kg) kg) kg) kg) kg) kg) 0 0 100 100 40 100 100100 100 0

The potency of conjugate 33 in this model of severe immunodeficiency wasalso evident based on body weights (Table 59). The lowest concentrationof conjugate providing full protection based on a mortality readout was0.1 mg/kg. At this dose level, the greatest average weight loss for thegroup was transient, and resulted in less than a 5% reduction (occurringon Day 2). Furthermore the difference in body weight for groups at the 1and 3 mg/kg dose levels showed less than a 2% difference on Day 14compared to uninfected mice.

Collectively this data demonstrates the potency of conjugate 33 byprotecting lethally challenged mice with single SC doses of conjugate aslow as 0.1 mg/kg. This was accomplished in a severe model ofimmunodeficiency with mice completely lacking T & B immune cells, whichare essential in clearing influenza infections. This data supports theuse of conjugate 33 to treat immune deficient patient populations.

TABLE 59 % Average body weight by day. (mg/kg). Data only shown untilthe first mortality within a group. hIgG1 Fc Baloxavir Conjugate 33 Day(3 (30 (10 (3 (3 (1 (0.3 (0.1 (0.03 post Vehicle mg/ mg/ mg/ mg/ mg/ mg/mg/ mg/ mg infection (PBS) kg) kg) kg) kg) kg) kg) kg) kg) /kg)Uninfected 0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0100.0 1 100.0 99.0 98.9 91.5 97.3 99.0 99.6 98.8 97.6 99.7 99.5 2 99.0100.8 99.1 97.3 100.1 100.1 99.9 100.7 95.8 98.6 99.8 3 94.0 93.0 97.096.4 97.4 100.0 98.9 97.6 97.0 96.7 98.8 4 93.0 89.7 99.1 95.9 99.0101.0 99.8 99.2 97.8 94.4 99.9 5 87.0 84.4 99.1 97.5 96.6 99.0 98.6 96.397.4 93.0 100.9 6 82.0 80.1 97.9 97.9 97.0 100.6 99.7 99.9 98.7 91.6100.2 7 78.0 98.6 97.0 97.2 101.9 99.6 99.6 98.1 87.0 101.0 8 98.6 96.495.4 100.9 98.2 99.2 98.3 82.2 100.4 9 99.6 95.1 93.1 100.6 98.4 100.097.0 100.7 10 96.0 95.9 90.5 98.8 98.8 98.5 95.8 100.2 11 97.3 97.3 99.099.3 98.3 96.0 99.7 12 98.7 95.8 99.1 99.4 98.1 96.3 100.4 13 98.6 96.898.4 99.0 97.9 97.3 99.7 14 97.9 95.9 99.3 99.3 98.1 97.8 100.5

Example 135. Efficacy of Conjugate 33 Subcutaneously Dosed AgainstInfluenza A/California/07/2009 (H1N1) Pdm in a Lethal Mouse Model

Conjugate 33 was evaluated against a lethal IAV H1N1 influenza infectionin female BALB/c mice (Charles River Laboratories, 6-8 weeks). Thechallenge virus (A/California/07/2009 (H1N1) pdm) is a pandemic isolatecapable of causing lethal infections in mice. The experiment comprised 6groups of 5 mice. At day 0, all mice were challenged with virus at 3×the LD₉₅ by intranasal inoculation in a volume of 30 μl, after beinganesthetized with a mixture of ketamine/xylazine (150 and 10 mg/kgrespectively). Mortality and body weights were recorded daily and anyanimal with a 20% loss of body weight was scored as a death.

Test groups received a single subcutaneous (SC) treatment of conjugate33, hIgG1 Fc control, or vehicle (PBS) 2 hours post viral challenge. Thestudy design and dose levels is summarized in Table 60.

TABLE 60 Study design for Influenza A/California/07/2009 (H1N1) pdmstudy Dose Dose volume Group Test article Route/Schedule (mg/kg) (ml/kg)# of mice 1 Vehicle SC, T + 2 hrs. na 10 5 2 hIgG1 Fc SC, T + 2 hrs. 110 5 3 conjugate 33 SC, T + 2 hrs. 1 10 5 4 conjugate 33 SC, T + 2 hrs.0.3 10 5 5 conjugate 33 SC, T + 2 hrs. 0.1 10 5 6 conjugate 33 SC, T + 2hrs. 0.03 10 5

As expected, mice receiving vehicle or the hIgG1 Fc control succumbed toinfection on Days 6-7 (Table 61). However, mice treated with conjugate33 were fully protected at 1 mg/kg, and nearly so at 0.3 (80% survival).Significant mortality with conjugate 33 was only seen at the lower doseconcentrations of 0.1 and 0.03 mg/kg.

TABLE 61 Percent survival by day. (mg/kg) hIgG1 Fc conjugate 33 (1.0(1.0 (0.3 (0.1 (0.03 Day Vehicle mg/kg) mg/kg) mg/kg) mg/kg) mg/kg) 0100 100 100 100 100 100 1 100 100 100 100 100 100 2 100 100 100 100 100100 3 100 100 100 100 100 100 4 100 100 100 100 100 100 5 40 20 100 10060 80 6 0 20 100 80 20 20 7 0 0 100 80 0 0 8 0 0 100 80 0 0 9 0 0 100 800 0 10 0 0 100 80 0 0 11 0 0 100 80 0 0 12 0 0 100 80 0 0 13 0 0 100 800 0 14 0 0 100 80 0 0

The potency of conjugate 33 was further supported by daily body weightmeasurements. As expected, mice treated with vehicle or hIgG1 Fcdemonstrated a steady drop in body weight until it exceeded 20%, atwhich time they were scored as a mortality (Table 62).

In contrast to control mice, mice receiving conjugate 33 at 1 mg/kg onlydemonstrated a transient drop in bodyweight of approximately 10%,peaking on Day 3 (Table 62). By both survival and body weightmeasurements conjugate 33 demonstrated robust protection from a lethalchallenge of Influenza A/California/07/2009 (H1N1) pdm with a single 1mg/kg dose administered SC. Activity against the clinically relevantpandemic strain used in this study supports the utility of conjugate 33in treating serious influenza infections.

TABLE 62 % Average body weight by day. (mg/kg). Data only shown untilthe first mortality within a group. hIgG1 Fc Conjugate 33 Day post (1.0(1.0 (0.3 (0.1 (0.03 infection Vehicle mg/kg) mg/kg) mg/kg) mg/kg)mg/kg) 0 100 100 100 100 100 100 1 99.5 97.9 96.8 96.7 98 98.7 2 97.197.2 99.5 99.2 98 98.4 3 86.5 86.0 89.7 89.6 87.6 88.8 4 80.3 80.3 91.688.5 81.3 81.9 5 76.2 76.6 92.9 91.0 76.8 77.9 6 94.7 90.5 7 94.2 8 95.49 98.0 10 96.8 11 98.8 12 97.7 13 100.1 14 100.5

Example 136. Efficacy of Conjugate 33 Intravenously (IV) Dosed AgainstInfluenza A/Puerto Rico/8/1934 (H1N1) in a Lethal Mouse Model of DelayedTreatment

Conjugate 33 was evaluated against a lethal influenza A (H1N1) infectionin female BALB/c mice (Charles River Laboratories, 6-8 weeks). Thechallenge virus (A/Puerto Rico/8/1934) is a mouse-adapted isolatecapable of causing lethal infections in mice. At day 0, all mice werechallenged with virus at 3× the LD₉₅ by intranasal inoculation in avolume of 30 μl, after being anesthetized with a mixture ofketamine/xylazine (150 and 10 mg/kg respectively). Mortality and bodyweights were recorded daily and any animal with a 20% loss of bodyweight was scored as a death.

The study design is detailed in Table 63, and consists of multiple arms.The control arm comprises vehicle (PBS) and hIgG1 Fc only groups, dosed24 hours after viral challenge (an uninfected group was also part ofthis arm). The second arm consisted of oseltamivir dosed at 4× itshumanized dose, with initiation of treatment delayed for 24, 48, or 72hours. The final 3 arms consisted of conjugate 33 administered as singleIV doses of 10, 3, or 1 mg/kg; each being dosed on the same schedule asthe oseltamivir arm above.

As expected, vehicle and hIgG1 Fc were not protective when dosed 24hours after viral challenge and resulted in complete mortality by Day 7.In our hands, oseltamivir, even at 4× the humanized dose (200 mg/kgcumulative dose) was only partially efficacious when dosing was delayed24 hours (Table 64; 40% survival). However, conjugate 33 was fullyprotective at all concentrations (10, 3, & 1 mg/kg) at the same 24 hourdose schedule.

When dosing was delayed a full 48 hours after viral challengeoseltamivir was no longer efficacious (0% survival) while conjugate 33was 80% protective at doses of 10 and 3 mg/kg. When dosing was delayeduntil 72 hours, only the 10 mg/kg dose of conjugate 33 demonstratedpartial protection (40%). The efficacy of conjugate 33 was also evidentbased on daily body weight measurements (Table 65). This was especiallysignificant in the T+24 hours groups where less than a 3% reduction wasobserved for any conjugate 33 group, which was transient and occurred onDay 1. In this study, conjugate 33 is more efficacious than oseltamivir,an approved treatment for influenza,

TABLE 63 Study design Dose Influenza A Test Route, First dose Dosevolume N Group strain Article Schedule (hours) (mg/kg) (ml/kg) (balb/c)1 A/PR/8/34 PBS IV, single T + 24 — 5 5 2 (H1N1) hIgG1 Fc IV, single T +24 10 5 5 3 3E2 Oseltamivir PO, bid × 5 T + 24 20 10 5 4 PFU/mouse daysT + 48 5 T + 72 6 AVC-076 IV, single T + 24 10 5 5 7 T + 48 8 T + 72 9AVC-076 IV, single T + 24 3 5 5 10 T + 48 11 T + 72 12 AVC-076 IV,single T + 24 1 5 5 13 T + 48 14 T + 72 15 Uninfected BW control

TABLE 64 % Survival by day. Test article/Dose/Time of treatmentinitiation Oseltamivir hIgG1 20 20 20 Conjugate 33 Conjugate 33Conjugate 33 Fc mpk, mpk, mpk, 10 10 10 3 3 3 1 1 1 Vehicle 10 bid × bid× bid × mpk, mpk, mpk, mpk, mpk, mpk, mpk, mpk, mpk, na mpk 5 5 5 singlesingle single single single single single single single T + T + T + T +T + T + T + T + T + T + T + T + T + T + Uninfected Day post 24 24 24 4872 24 48 72 24 48 72 24 48 72 na infection hrs. hrs. hrs. hrs. hrs. hrs.hrs. hrs. hrs. hrs. hrs. hrs. hrs. hrs. na 0 100 100 100 100 100 100 100100 100 100 100 100 100 100 100 1 100 100 100 100 100 100 100 100 100100 100 100 100 100 100 2 100 100 100 100 100 100 100 100 100 100 100100 100 100 100 3 100 100 100 100 100 100 100 100 100 100 100 100 100100 100 4 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 5100 100 100 100 100 100 100 100 100 100 100 100 80 100 100 6 60 100 100100 100 100 100 60 100 100 80 100 80 80 100 7 0 0 100 100 20 100 100 40100 80 0 100 60 40 100 8 0 0 100 100 0 100 80 40 100 80 0 100 40 0 100 90 0 60 60 0 100 80 40 100 80 0 100 40 0 100 10 0 0 40 0 0 100 80 40 10080 0 100 40 0 100 11 0 0 40 0 0 100 80 40 100 80 0 100 40 0 100 12 0 040 0 0 100 80 40 100 80 0 100 40 0 100 13 0 0 40 0 0 100 80 40 100 80 0100 40 0 100 14 0 0 40 0 0 100 80 40 100 80 0 100 40 0 100

TABLE 65 Body Weight Data (gm). mpk = mg/kg. Data only shown until thefirst death within a group. Test article/Dose/Time of treatmentinitiation Oseltamivir hIgG1 20 20 20 Conjugate 33 Conjugate 33Conjugate 33 Fc mpk, mpk, mpk, 10 10 10 3 3 3 1 1 1 Vehicle 10 bid × bid× bid × mpk, mpk, mpk, mpk, mpk, mpk, mpk, mpk, mpk, Day na mpk 5 5 5single single single single single single single single single post T +T + T + T + T + T + T + T + T + T + T + T + T + T + Uninfected infec- 2424 24 48 72 24 48 72 24 48 72 24 48 72 na tion hrs. hrs. hrs. hrs. hrs.hrs. hrs. hrs. hrs. hrs. hrs. hrs. hrs. hrs. na 0 100 100 100 100 100100 100 100 100 100 100 100 100 100 100 1 99.3 98.8 96.4 97.5 98.2 97.697.7 98.5 97.1 98.3 98.7 98.3 99.4 99.4 99.5 2 103.9 101 100.7 101.2102.6 102.1 101.7 100.1 102 102.5 99.4 104.2 102 101.4 101.7 3 96.9 97.495.5 97.5 95.6 102.3 96.2 95.1 100.6 95.9 97.6 102.3 96.9 98.6 105.8 488.7 90.1 92.6 89.7 87.7 102.3 88.4 88.8 100 87.8 88.6 100 89.2 89.7 1055 81.7 83.9 92.2 88.6 81.6 101.5 88.1 82.7 99 85.3 81.7 101.2 83.7 83.2105.5 6 77.3 79.3 91.8 86 78.6 102.6 91.1 80.4 101.4 87.1 77.5 103.378.7 104.9 7 87.8 81 101.9 91.1 102.3 86.1 102.1 103.5 8 83.5 75.8 101.591.3 101.1 103.2 102.9 9 81.1 102.2 102.1 104.1 102.9 10 102.5 101.6 105103.3 11 102.4 102.1 103.9 104.3 12 100.9 101.6 103.4 102.5 13 101 101.8103.5 102.5 14 102.2 103.6 104 103.1

Example 137. Synthesis of Conjugate 38 (Int-73), Conjugate 39 (Int-74),Conjugate 40 (Int-75), and Conjugate 41 (Int-76)

A 15-ml sterile centrifuge tube is charged with sodium ascorbate (68.3mg, 0.345 mmol), BTTAA (11.9 mg, 0.0276 mmol), product from Examples 114(Int-73), 115 (Int-74), 116 (Int-75), or 117 (Int-76) (0.00953 mmol) andPBS 7.4 (1 ml). The reagents were vortexed until homogeneous then mixedwith azido Fc (50 mg, 0.0008624 mmol, described in Example 124, SEQ IDNO: 64) followed by a solution of CuSO4 (1.1 mg, 0.0069 mmol) in water(0.5 ml). The mixture is rotated for 12 hours then purified by affinitychromatography over a protein A column, followed size exclusionchromatography. Conjugates are characterized by Maldi TOF analysis (DARtypically=4.5). Yields are typically 50%.

Example 138. Synthesis of Int-79

Step a

Zanamavir-ether-acid (0.90 g, 1.43 mmol, Example 31) and N-methylmorpholine (0.23 mL, 2.14 mmol) were dissolved in THF (35 mL) and cooledto 0° C. (ice water bath) under an atmosphere of nitrogen. Isobutylchloroformate (0.24 mL, 1.85 mmol, in 2 mL DCM) was added dropwise, byway of syringe over a 5 minute period. The mixture was stirred at 0° C.for 30 minutes then 15 min at ambient temperature, and then cooled to 0°C. Sodium borohydride (540 mg, 14.3 mmol, dissolved in 5 mL of methanol)was added, dropwise over 5 minutes. The reaction was stirred for 15minutes at which point all starting material had been consumed (byLC/MS). A few drops (˜1 mL) of glacial acetic acid was added to acidifythe mixture (pH˜5). Dilute with ethyl acetate and water and extract intoethyl acetate (3×). The organic layer was washed with brine, and theorganic extracts were dried over sodium sulfate, and concentrated on therotary evaporator. The crude material was purified by silica gelchromatography (loaded on celite first) (0-10% methanol in DCM, 30 min).Yield 0.66 g, 75%. Ion found by LCMS: [M+H]+=617.2.

Step b

To a stirring mixture of alcohol (0.66, 1.1 mol, in 20 mL CH₂Cl₂), wasadded triethylamine (0.30 mL, 1.3 mmol). The mixture was cooled to 0° C.(ice-water bath) under 1 atmosphere of nitrogen and mesyl chloride (150mg, 1.3 mmol) was added, dropwise over 5 minutes by way of syringe. Theice bath was removed and the reaction was stirred for 45 minutes. Themixture was diluted with saturated, aqueous sodium bicarbonate,extracted into DCM (3×). The combined organic extracts were washed withbrine, dried over sodium sulfate and concentrated on the rotaryevaporator. Ion found by LCMS: [M+H]+=695.2. The intermediate was takento the next step without purification.

The zanamvir mesylate was stirred in DMF at 80° C. with 3 eq of sodiumazide for 5 hours. The mixture was diluted with water, extracted intoDCM (3×). The combined organic extracts were washed with brine, driedover sodium sulfate and concentrated. Ion found by LCMS: [M+H]+=695.2.The azide was taken to the next step without purification.

The zanamavir azide (670 mg, 1.04 mmol) was stirred in methanol (20 mL)in the presence of Lindlar catalyst (300 mg) under 1 atmosphere ofhydrogen gas for 12 hours. The mixture was filtered through celite andconcentrated to afford the title compound as a clear oil. The amine wastaken to the next step without purification. Yield 0.37 g, 54%, 3 steps.Ion found by LCMS: [M+H]+=616.2.

Step c

Methyl 3-(benzylamino)propanoate (1 g, 5.2 mmol), methyl4-bromobutanoate (1.2 g, 6.2 mmol), and diisopropylethylamine (1.34 mL,7.8 mmol) were stirred in DMF (5 mL) at 65° C. for 4 hours. Most of thesolvent was removed by rotary evaporator and the crude material waspurified by silica gel chromatography (Isco, 0 to 9% methanol in DCM, 30minute gradient) to afford the benzyl protected intermediate as a clearoil. The benzyl protected intermediate was stirred in methanol (20 mL)under 1 atmosphere of hydrogen gas in the presence of 20% palladiumhydroxide on carbon (300 mg) for 12 hours. The mixture was filteredthrough celite and concentrated to afford the title compound as a clearoil. Yield 0.72 g, 69%. Ion found by LCMS: [M+H]+=204.2.

Step d

The step-c product (0.70 g, 2.16 mmol), propargyl PEG-4 acid (0.63 mg,2.38 mmol) HATU (1.26 g, 3.24 mmol) in DMF (2 mL) were stirred at roomtemperature following by addition of DIEA (1.15 mL, 6.49 mmol). Thereaction mixture was stirred for 2 hours then purified by reverse phaseliquid chromatography (Isco, 5 to 50% acetonitrile and water with 0.1%TFA as modifier). Yield 840 mg, 87%. Ion found by LCMS [M+H]+=446.2.

Step e

A solution of step-d product (840 mg, 1.85 mmol) and LiOH (113.1 mg,4.72 mmol) in H₂O:MeOH (1:2, 9 mL) was stirred at room temperature for 2hours. Then the reaction was acidified with TFA. The resulted solutionwas concentrated then purified by reverse phase liquid chromatography(Isco, 0% to 20% acetonitrile and water). Yield 454.5 mg, 59%. Ion foundby LCMS [M+H]+=418.2.

Step f

To a solution of step-b product (334.7 mg, 0.52 mmol), step-e product(102 mg, 0.24 mmol) and HATU (273.25 mg, 0.704 mmol) in anhydrous DMF (3mL) at room temperature was added DIEA (217 mg, 16.4 mmol). Theresulting mixture was stirred for 1 hour then was purified by RPLC (20%to 100% methanol and water without modifier). Yield 206.5 mg, 55%. Ionfound by LCMS [(M+2H)/2]+=806.8.

Step g

The step-f product (206.5 mg, 0.128 mmol) and TFA (3 mL) in CH₂Cl₂ (5mL) was stirred at room temperature overnight, then concentrated underreduced pressure. The resulting residue was purified by semi-preparativeHPLC (0% to 30% acetonitrile and water with 0.1% TFA as modifier). Yield144.5 mg, 69%. Ion found by LCMS [(M+2H)/2]+=606.8.

Step h

To a solution of the step-g product (144.5 mg, 0.119 mmol) in MeOH (9mL) and water (3 mL) was added LiOH (18 mg, 0.75 mmol). The resultingsolution was stirred at room temperature for 1 hour, then acidified withTFA and concentrated under reduced pressure. The residue was purified bysemi-preparative HPLC (0% to 25% acetonitrile and water, using 0.1% TFAas modifier). Yield 45 mg, 28%. Ions found by LCMS [(M+2H)/2]+=566.8.

Example 139. Synthesis of Conjugate 42

A solution of azido functionalized Fc (50 mg, 5.4 mL, 0.859 μmol,Example 124, SEQ ID NO: 18) was added to a 10 mL centrifuge tubecontaining alkyne derivatized small molecule (7.87 mg, 0.057 mmol,Example 138). After gently shaking to dissolve all solids, the mixturewas added to 3 mL premixed solution of L-ascorbic acid sodium (0.68 mg,0.34 mmol, 0.25 M), copper (II) sulfate (1.1 mg, 0.0069 mmol, 0.005 M)and BTTAA (11.8 mg, 0.027 mmol, 0.02 M) in PBS 7.4 buffer. The resultingsolution was gently rotated overnight. It was purified by affinitychromatography over a protein A column, followed by size exclusionchromatography (See general conjugate purification protocol). Maldi TOFanalysis of the purified final product gave an average mass of 63623 Da(DAR 3.8). Yield 36.01 mg, 72%.

Example 140. Synthesis of Int-80

Step a

To a solution of the p-nitrophenyl carbonate of Zanamivir (0.3 g. 0.4mmol, Example 103) in anhydrous dichloromethane (5 ml) was added apropargyl-PEG4-methylamine (0.11 g, 0.44 mmol) and DIPEA (0.14 ml, 1.0mmol) in anhydrous DMF (5 ml). The reaction was stirred at roomtemperature overnight, then concentrated and purified by flashchromatography eluting with 0% to 10% methanol/dichloromethane. Yield0.28 g, 81%. Ions found by LCMS: (M+H)⁺=858.4, (M-Boc)+H⁺=758.4.

Step b

Product from the previous step (280 mg, 0.2 mmol) was dissolved into 2ml MeOH and 2 ml THF, then treated with a solution of lithium hydroxide(24 mg, 1 mmol) dissolved in 2 ml water. The reaction was stirred for 10min at room temperature at which time HPLC showed the reaction wascomplete. The pH of the reaction was adjusted to the value of 5 to 6 byusing Amberlite IRN-77 ion exchange resin, then filtered to remove theresin. The crude filtrate was evaporated to dryness under a vacuum andused in the next step with purification, and the yield was quantitative,Ion(s) found by LCMS: (M+H)⁺=844.4, (M-Boc+H)⁺=744.4.

Step c

The step-b product was dissolved into 2 ml dichloromethane and 2 ml TFA,and stirred at room temperature. The progress of the reaction wasmonitored by LCMS. After the completion of the reaction (6 h), thesolution was stripped to dryness and then dissolved in 2 ml water and 2ml acetonitrile. The resulting solution was stirred for another 2 hourat room temperature at which time LCMS show complete deprotection of theacetonide protecting groups. This mixture was concentrated and purifiedby reverse phase liquid chromatography (RPLC) using an Isco CombiFlashliquid chromatograph eluted with 5% to 40% acetonitrile/water with 0.1%TFA as the modifier. Yield 180 mg, 78.0%. Ion(s) found by LCMS:(M+H)⁺=604.2.

Example 141. Stability Data for Int-80 Compared to Int-4

Int-80 (Example 140) and Int-4 (Example 13) were solubilized to 10 mg/mLin deionized water and then diluted 1:10 into 1×PBS to a finalconcentration of 1 mg/mL. Samples were incubated at 37° C. or 60° C. for1 week. A 25 uL aliquot was diluted into 75 uL of water for HPLCanalysis. Using a Waters Acquity H-Class UPLC with a Phenomenex BiozenPS-C18 column (150×2.1 mm, 1.6 um) a gradient of 0.1% formic acid inwater to 0.1% formic acid in acetonitrile was run as follows: 5% B for0-1 min, 5-20% B from 1-20 min. Detection was at 240 nm using a diodearray detector, Int-80 had less than 1% degredation over a week at 60°C., while Int-4 showed 15% degredation over the same time period (FIG.66)

Example 142. Synthesis of Conjugate 43

Preparation of the Click reagent solution: 0.0050M CuSO4 in PBS×1 buffersolution: 10.0 mg CuSO4 was dissolved in 12.53 mL PBS×1, than took 10.00mL this CuSO4 solution and added 86.1 mg BTTAA and 495.3 mg Na Ascorbateto give the Click reagent solution (0.0050M CuSO4, 0.020M BTTAA and0.25M Sodium Ascorbate).

A solution of azido functionalized Fc (78.0 mg, 4.535 mL, 1.35 μmol,Example 124, SEQ ID NO: 64) was added to a 15 mL centrifuge tubecontaining alkyne derivatized small molecule (13.2 mg, 8.88 umol,Int-80, Example 140). After gently shaking to dissolve all solids, themixture was added with 2.153 10 mL of above Click reagent solution of(L-ascorbic acid sodium, 0.25 M, 106.6 mg, 0.538 mmol, copper (II)sulfate 0.0050M, 1.72 mg, 0.0107 mmol, and BTTAA 0.020M, 18.5 mg, 0.0431mmol). The resulting mixture was gently rotated for 6 hours at ambienttemperature. It was purified by affinity chromatography over a protein Acolumn, followed size exclusion chromatography (See conjugatepurification protocol). Maldi TOF analysis of the purified final productgave an average mass of 64,012 Da (DAR=7.0). Yield 50.3 mg, 62% yield.

Example 143. Synthesis of Int-81

Step a

To a well-stirred solution of N-Boc-N-Me-glycinol (3.5 g, 20 mmol) inDMSO (20 mL) cooled with an ice-water bath was add allyl bromide (3.6 g,30.0 mmol), followed by finely ground KOH powder (3.5 g, 30.0 mmol) over15 minutes. The resulting solution was stirred for overnight at roomtemperature. The resulting mixture was partitioned between 5% aq. HOAc(50 mL) and ethyl acetate (200 ml). The organic layer was separated,washed with brine, dried with sodium sulfate, filtered, andconcentrated, then purified by flash chromatography eluting with 10% to80% ethyl acetate/hexane. Yield of product 4.1 g, 95%. Ion(s) found byLCMS: M+H=216.3.

Step b

Ozone was bubbled through a solution of the compound from step a (8.0 g,37 mmol) in MeOH (50 mL) and DCM (50 ml) at −78° C. until the appearanceof a light blue color. Unreacted ozone was removed by bubbling withoxygen for 10 minutes before the addition of NaBH₄ (1.8 g, 40 mmol) insmall portion over 10 minutes. After all NaBH₄ was added the mixture wasgradually warmed to room temperature. The resulting solution waspartitioned between ethyl acetate (100 ml) and brine (50 ml). Theorganic layer was separated, washed with brine, dried with sodiumsulfate, filtered, concentrated to an oil, and then purified by flashchromatography eluting with 10% to 80% ethyl acetate/dichloromethane.Yield of product 5.0 g, 62%. Ion(s) found by LCMS: M+H=220.2.

Step c

To a solution of the product (4.4 g, 20 mmol) from the previous step andCBr₄ (10.0 g, 30.0 mmol) in DCM (50 mL) at 0° C. was added PPh₃ (8.0 g,30 mmol) slowly over 15 minutes (exothermic). During the course of theaddition the internal temperature was kept below 30° C. After additionof PPh₃ the reaction was stirred overnight at room temperature. Thecrude reaction was concentrated to an oil then purified by normal phasechromatography, eluting with 10% ethyl acetate/hexanes to 80% ethylacetate/hexanes. Fractions containing oil droplets on the inside of thecollection tubes were combined and concentrated to a colorless oil. 4.0g, 70.5%. Ion(s) found by LCMS: M+H=282.1.

Step d

A solution of the step c product (4 g, 14 mmol), benzylamine (0.60 g,5.7 mmol), and K₂CO₃ (2.35 g, 17 mmol) in DMF (20 mL) were heated in anoil bath at 75° C. for 8 h. The mixture was filtered, concentrated, andpurified by RPLC (5% ACN/water to 100% ACN). Yield 2.1 g, 72.7%.

Step e

To a solution of the step-d product (1.3 g, 2.0 mmol) dissolved inCHCl₃/EtOH (1:20, 20 mL) was added 20% Pd(OH)₂/C (0.50 g). The reactionwas stirred overnight under hydrogen from a balloon at ambienttemperature. The reaction mixture was filtered through a Celite pad,then concentrated by way of rotary evaporator and carried to thesubsequent step without further purification.

Step f

The step-e product was dissolved in 10 mL of DMF, then treated withpropargyl PEG₄ acid (0.52 g, 2.0 mmol), EDCI (0.6 g, 3.0 mmol), HOAt(0.45 g, 3 mol) and Hunig's base (0.7 mL, 5.0 mmol) at room temperature.The reaction mixture was stirred for four hours, then concentrated andpurified by RPLC (10% ACN/water to 60% ACN/water). Yield 0.43 g, 65% fortwo steps. Ions found by LCMS: [M-Boc+H]⁺=562.4, [M+H]⁺=662.4.

Step g

The step-d product (70 mg, 0.1 mmol) was treated with TFA (2 mL) for 2hours at room temperature. TFA was removed by rotary evaporation, andthe remaining oil was further dried under high vacuum for 12 h to givethe desired product as bis-TFA salt. Yield was quantitative. Ion foundby LCMS: [M+H]⁺=462.4.

Step h

To a solution of the nitrophenyl carbonate described in the Example 109(0.72 g. 0.95 mmol) in anhydrous DMF (5 ml) was added a mixture of thestep g diamine (0.3 g, 0.43 mmol, added in portions over 30 minutes) andDIPEA (0.28 ml, 2 mmol) in anhydrous DMF (20 ml). The reaction wasstirred at room temperature overnight, then concentrated and purified byflash chromatography eluting with 0% to 10% methanol/dichloromethane.Yield 0.63 g, 86%. Ions found by LCMS: [(M+2H)/2]⁺=844.4,[(M-Boc+2H)/2]⁺=794.4, [(M-2Boc+2H)/2]⁺=744.4.

Step i

Product from the previous step (600 mg, 0.35 mmol) was dissolved into 5ml MeOH and 5 ml THF, then treated with a solution of lithium hydroxide(48 mg, 2 mmol) dissolved in 2 ml water. The reaction was stirred for 10min at room temperature at which time HPLC showed the reaction wascomplete. The pH of the reaction solution was adjusted to a value of 5to 6 by using Amberlite IRN-77 ion exchange resin, then filtered toremove the resin. The crude product was evaporated to dryness by rotaryevaporation and used in the next step with purification, Ion(s) found byLCMS: [(M+2H)/2]⁺=829.9, [(M-Boc+2H)/2]⁺=779.4, [(M-2Boc+2H)/2]⁺=729.4.

Step j

The product from step-i was dissolved into 5 ml dichloromethane and 5 mlTFA, and stirred at room temperature. The progress of the reaction wasmonitored by LCMS. After the completion of the reaction (6 h), thesolution was stripped to dryness and then dissolved in 4 ml water and 4ml methanol. The resulting solution was stirred for another 2 hour atroom temperature at which LCMS show complete deprotection of theacetonide protecting groups. This mixture was concentrated and purifiedby reverse phase liquid chromatography (RPLC) using an Isco CombiFlashliquid chromatograph eluted with 5% to 40% acetonitrile/water with 0.1%TFA as the modifier. Yield 380 mg, 71.0%. Ion(s) found by LCMS:[(M+2H)/2]⁺=589.8, [(M+3H)/3]⁺=392.5.

Example 144. Synthesis of Int-82

The title compound was prepared analogously to Example 143, Int-81,where the N-Boc-N-Me-glycinol was substituted withN-Boc-N-ethyl-glycinol in the step a. Ion(s) found by LCMS:[M/2]+1=603.8, [M/3]+1=402.9.

Example 145. Synthesis of Int-83

Step a

The CBZ-protected-amino-peg1-bromide (7.6 g, 25.2 mmol), benzylamine(1.1 g, 10.1 mmol), and potassium carbonate (2.8 g, 2.8 mmol) werestirred in DMF (10 mL) at 60° C. for 12 hours. The solution wasconcentrated on the rotary evaporator and purified by silica gelchromatography (0-10% methanol in DCM, 30 minute gradient) to afford theproduct as a clear viscous oil. Yield 3.2 grams, 57%. Ion found by LC/MS[M+H]+=550.2.

Step b.

The product of step a (1.9 g, 3.5 mmol) was dissolved in THF (20 mL) andcooled to 0° C. by way of an ice/water bath under an atmosphere ofnitrogen. LAH (6.9 ml, 13.8 mmol, 2M in THF) was added dropwise by wayof syringe over a period of 10 minutes. The mixture was stirred atreflux for 2 hours then cooled to 0° C. byway of an ice/water bath. 1 mLof water was added, dropwise followed by the dropwise addition of 1 mLof aqueous (15% by weight) NaOH solution. 3 mL of water was added andthe mixture was stirred for 15 minutes at which time 2 g of magnesiumsulfate was added. The mixture was stirred for 10 minutes then filteredthrough celite, washed with 2 additional 10 ml portions of THF and thecombined filtrates were concentrated by the rotary evaporator. Theresidue was taken up in acetonitrile (20 mL), triethylamine (1.4 g, 13.8mmol) and boc anhydride (3.0 g, 13.8 mmol) were added. The mixture wasstirred for 45 minutes, concentrated and purified by reversed phase HPLC(5-95% acetonitrile/di water, 0.1% TFA modifier, 30 minute gradient).Yield 1.4 g, 79%. Ion found by LC/MS [M+H]+=510.2.

Step c

The product from step b. (1 g, 1.9 mmol) of this example was stirred inmethanol (25 mL) in the presence of palladium hydroxide (200 mg) underan atmosphere of hydrogen for 2 hours. The mixture was filtered throughcelite and concentrated to afford the product as a clear oil which wasused with no further purification. Yield 0.73 g, 89%. Ion found by LC/MS[M+H]+=420.4.

Step d

The product from step c (0.73 g, 1.7 mmol), propargyl-peg4-tosylate(0.91 g, 2.4 mmol), and diisopropylethylamine (0.76 g, 5.9 mmol) werestirred in DMF (5 mL) at 85° C. for 4 hours. The mixture wasconcentrated on the rotary evaporator, then purified by reversed phaseHPLC (5-95% acetonitrile/DI water, 0.1% TFA modifier, 30 minutegradient) to afford the product as a clear viscous oil. Yield 0.89 g,82%. Ion fond by LC/MS [M+H]+=634.4.

Step e

The product from step d. (0.89 g, 1.4 mml) was stirred in 4N HCl (indioxane) for 45 minutes at ambient temperature. The mixture wasconcentrated on the rotary evaporator and azeotroped (3×) with benzene.The resulting residue was taken up in DI water (15 mL) frozen andlyophilized to afford the product as a clear oil, bis-HCl salt. Yield0.65 g, 91%. Ion found by LC/MS [M+H}+=434.4.

Step f

The remaining four steps in the synthesis of this compound wereanalogous to those used in the synthesis of Example 143, Int-81. Ion(s)found by LCMS: (M+2H)/2=575.8

Example 146. Alternate Synthesis of Int-83

The product described in Example 145, Int-83, can be preparedalternatively using the above reaction scheme by a person skilled in theart using methods described in this patent.

Example 147. Serial Passage Experiments for Selection of ResistantInfluenza Viruses

To evaluate the potential for development of drug resistant mutant viralstrains under selective pressure with viral inhibitors, serial passagestudies were conducted with Conjugates 6 and 33, versus oseltamivir andbaloxavir comparators. Serial passage studies were conducted usingeither A549 or MDCK cells. Passages were conducted as follows: 150,000A549 or MDCK cells were seeded per well (12-well) in 500 μl DMEM 10%FBS, 1% PS, 1% NaPyr and 1% HEPES and incubated for approximately 24hours. Once cells reached approximately 80% confluency, they were washedonce with PBS and incubated for 2 hours in the presence of compounds orPBS alone under normal culture conditions. Test article concentrationswere optimized as required for maximum virus inhibition, whilemaintaining enough virus production for subsequent passages.Concentrations of test articles used in the serial passage experimentsare shown in FIGS. 67 and 68. Cells were then infected at an MOI of 0.01or 0.05 (MDCK and A549 cells, respectively; 150 ul diluted in infectionbuffer) for 1 hour at room temperature in a buffer containing PBS,Bovine Albumin 35% and Ca²⁺/Mg²⁺, followed by removal of inoculum andwashing of cells once with DMEM 1% PS, 1% NaPyr and 1% HEPES (No FBS).Cells were then Incubated for 24 hours in the presence of test articlesdiluted in DMEM 1% PS, 1% NaPyr, 1% HEPES and 1 μg/ml TPCK-treatedtrypsin. After incubation, viral supernatants were collected, and cellsand debris were removed by centrifugation (5 min, 4° C., 1,400×g).Supernatants were then used to:

i Quantify viral titer by plaque assay

ii Conduct a hemagglutination assay to determine if the virus escapedcompound inhibition

iii Re-infect freshly seeded A549 or MDCK cells in presence ofcompounds.

The process was repeated for 10 passages, or until resistance wasobserved for the oseltamivir and baloxavir controls. Once increasedtiters in the presence of drugs is detected for two consecutivepassages, the viruses may be plaque purified. Following plaquepurification, all 8 genome segments may be sequenced and compared to PBStreated control virus to detect escape mutations. Summaries of twodifferent serial passage experiments are shown in FIGS. 67 and 68. Inthe experiment summarized in FIG. 67, Conjugate 6 was compared tooseltamivir and baloxavir using A549 cells infected withA/California/04/09/H1N1 pdm. Conjugate 6, baloxavir and oseltamivir wereused at 0.5 nM, 0.5 nM and 200 nM, respectively. No increases in viraltiter were observed for Conjugate 6 through the course of 11 passages(suggesting no emergence of resistant mutants), while baloxavir andoseltamivir titers increased to levels similar to those observed in thePBS control after passages 5 and 11, respectively. In the experimentsummarized in FIG. 68, Conjugates 6 and 33 were compared to oseltamivirand baloxavir using MDCK cells infected with A/WSN/1933 H1N1. Conjugate6, Conjugate 33, baloxavir and oseltamivir were used at 4 nM, 2 nM, 4 nMand 50 nM, respectively. No increases in viral titer were observed forConjugates 6 or 33 through the course of 10 passages (suggesting noemergence of resistant mutants), while baloxavir and oseltamivir titersincreased to levels similar to those observed in the PBS control afterpassages 5 and 10, respectively.

1. A conjugate described by formula (D-I):

wherein each of A₁ and A₂ is zanamivir; E comprises an Fc domainmonomer; L is a linker covalently attached to each of A₁, A₂, and E,wherein L is covalently attached to each of A₁ and A₂ at position C7 orposition C9 of zanamivir; n is 1 or 2; and T is an integer from 1 to 20;or a pharmaceutically acceptable salt thereof.
 2. The conjugate of claim1, wherein each L is covalently attached to the nitrogen atom of asurface exposed lysine of E.
 3. The conjugate of claim 1, wherein each Lis covalently attached to the sulfur atom of a surface exposed cysteineof E.
 4. The conjugate of claim 1, wherein n is 2, and each E dimerizesto form an Fc domain.
 5. The conjugate of claim 1, wherein each Ecomprises a serine substitution at C220, numbered according to the KabatIndex.
 6. The conjugate of claim 1, wherein T is 1, 2, 3, 4, 5, 6, 7, 8,9, or
 10. 7. A population of conjugates of claim 1, wherein the averagevalue of T is 1 to
 5. 8. A method for the treatment of a subject havinga viral infection or presumed to have a viral infection, the methodcomprising administering to the subject an effective amount of aconjugate of claim
 1. 9. A method for the prophylactic treatment of aviral infection in a subject in need thereof, the method comprisingadministering to the subject an effective amount of a conjugate of claim1.